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 regulated by covalent modification. In whole cells containing high levels 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-glutamyltransferase 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 ammonia-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 bacteria such as E. coli addition of ammonium chloride to a nitrogen-limited culture results in covalent modification of GS by 30 0- adenylylation (4). Adenylylation decreases the biosynthetic activity ofE. coli GS and alters the pH profile for transferase activity (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. In- cubation of S. cattleya extracts with SVPDE, known to cleave FIG. 1. Kinetics of GS inactivation in whole cells. S. cattleya cells the AMP moiety ofadenylylated E. coli GS, had a significant ef- growing in glucose/glutamate minimal medium were assayed for GS transferase activity by taking 80-pl samples and adding them to cold fect on the activity in the extract from ammonia-shocked cells assay mixture containing cetyltrimethylammonium bromide at 100 (Fig. 2). GS transferase activity increased nearly to that of the gg/ml. At 0 min ammonium chloride was added (to 20 mM) and sampling was continued. After all samples were taken transferase assays * Wax, R. & Snyder, L. (1980) Annual Meeting of the American Society were run at 370C for 20 min. for Microbiology, Miami Beach, FL, p. 187 (abstr.). Downloaded by guest on October 1, 2021 Biochemistry: Streicher and Tyler Proc. Nati. Acad. Sci. USA 78 (1981) 231

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FIG. 3. Two-dimensional O'Farrell gels (16) of ammonia-shocked and unshocked cell extracts. Samples of the ammonia-shocked and unshocked crude extracts used for the experiment described in Fig. 2 were analyzed. The basic end of the- gel is on the left, the acidic end on the right. (A) Control (unshocked) extract. (B) enlarged section ofA (boxed area). (C) Ammonia-shocked cell extract. (D) enlarged section of C (boxed area). Arrows point to the nonadenylylated GS subunit (the more basic~~~~~~..of the pair). -control extract. There was no alteration ofactivity in the control confirm this interpretation we purified S. cattleya GS and ..extract~treated with SVPDE or an increase in activity ofthe ex- showed that the protein pair in question represented the inter- tract of ammonia-shocked cells in the absence of SVPDE. In convertible forms ofthe GS subunit (unpublished). We obtained similar experiments we monitored forward biosynthetic activity additional evidence that the change in isoelectric point was due and found that it also significantly increased after SVPDE treat- to the phosphodiester linkage by incorporating inorganic ment (data not shown). We conclude from these results that S. [32P]phosphate into GS. In this experiment we added cattleya GS is modified after ammonia shock to an inactive form [32P]phosphate to a mid-logarithmic culture and harvested cells through a covalent linkage involving a phosphodiester bond. before and after ammonia shock. Extracts were prepared and The addition ofa phosphodiester group to a protein would be analyzed on two-dimensional gels. When we compared the expected to alter its charge and therefore the isoelectric point. stained gels with the autoradiographs we observed that 32p was We analyzed extracts from control and ammonia-shocked cells incorporated only into the more acidic protein of the GS pair. by using the O'Farrell two-dimensional polyacrylamide gel The amount of32p incorporated into the GS protein greatly in- electrophoresis technique (16) to separate proteins by isoelec- creased after ammonia shock, and all the radioactivity in the GS tric point as well as molecular weight. The only noticeable dif- protein was removed after treatment with SVPDE. 32p was also ference in the protein profiles of the two extracts was found in incorporated into several other proteins. A few ofthese appear the amounts oftwo proteins differing only in charge (Fig. 3). In to contain phosphodiester bonds, because the radioactivity was the control extract the more basic protein ofthe pair was present removed by SVPDE incubation. We do not know the identity of in a much higher amount than the more acidic one. However in these proteins. the extract from ammonia-shocked cells the situation was re- Modification of S. cattleya GS in Crude Extracts. The addi- versed: the more acidic protein was present in a much higher tion ofATP to crude extracts caused a rapid loss ofGS transferase amount than the more basic one. After treatment of the latter activity (Fig. 4). ATP was specifically required for the modifi- extract with SVPDE the pattern reversed and the more acidic cation reaction; UTP, CTP, and GTP were all inactive. We were protein was barely detectable-(gel not shown). The change in able to demonstrate a stimulatory effect ofglutamine on the rate relative amounts of these two proteins is consistent with their of modification (Fig. 4B) when we used dialyzed extracts. being the modified and unmodified forms ofthe GS subunit. To Crude extracts prepared by our standard procedures required Downloaded by guest on October 1, 2021 232 Biochemistry: Streicher and Tyler Proc. Nad Acad. Sci. USA 78 (1981) effect ofglutamine, and the inhibitory effect ofa-ketoglutarate on GS modification are very similar to the conditions for adenylylation of E. coli GS. We modified the GS in crude extracts in the presence ofeither [a-32P]ATP or [adenine-3H]ATP. The inactivated GSs present in both extracts were analyzed by using nondenaturing and NaDodSO4 gel electrophoresis. The Na- DodSO4 gel demonstrated the incorporation of both [32p]- and [3H]ATP into the GS subunit. This was confirmed with a non- 70 denaturing gel. In this case we also found that modification of GS did not result in a significant change in the native enzyme .~60 60- structure. The position of the labeled GS coincided with the < stained GS and had mobility identical to that ofpurified active 50 m50 GS. Therefore it is clear that adenylylation ofGS, while causing a drastic loss ofenzymatic activity, did not also causeaggregation FI 40 -P and u 40n oA or disassembly of the native enzyme. These labeling studies provide strong evidence that S. cattleya GS is modified by 30 30 V adenylylation.

20 %20- o~~ ~ ~~~~Tminn) DISCUSSION S. cattleya is a Gram-positive filamentous spore-forming bacte- ~~~~~U 0 rium (11). In most respects it is quite different from the enteric 0 jZR I bacteria. We have shown here, however, that with respect to 0 510O1520'25 30 0 510 152025 30 glutamine synthetase there is a remarkable similarity. In a pre- Time (min) liminary report, Wax and Snyder* found that an ammonia shock ofS. cattleya cells growing on lysine as the source ofnitrogen led FIG. 4. ATP and glutamine dependence ofCS inactivation in crude to a rapid loss ofGS transferase activity. Our experiments dem- extracta. A crude, extract was prepared from cells grown on glucose/ onstrate that when S. cattleya cells growing under nitrogen-lim- glutamate medium and was -dialyzed overnight against 500 vol of bufferA. Then 0.2 ml ofcrude extract was added to 0.2 ml ofprewarmed iting conditions were exposed to ammonium chloride a rapid adenylylation reaction mixture (15). At the indicated times-5-,ul sam- modification ofGS occurred, with the concomitant loss ofall GS ples were taken, added to cold transferase reaction mixtures, and, at activities. We found that full activity was recovered by treat- the completion ofthe sampling, assayed at 370C for 10 min. (A) Effect ment of crude extracts with SVPDE. This suggested that the ofvarying the ATP concentration. ATP concentrations were: o, 0 mM; modification of GS involved a phosphodiester linkage. Radiola- *, 1 mM; *, 2 mM; *, 3 mM; A, 5 mM; n, 10 mM. (B) Effect ofvarying beling experiments also established that the adenine moiety of Glutamine concentrations were: e, 0 the glutamine concentration. ATP was covalently incorporated into GS subunits, leading us to mM; A, 2.5 mM; m,5- mM; o, 10mM; o, 30mM. conclude that the enzymatic activity of S. cattleya GS is 'regulated by adenylylation. Adenylylation of GS in crude extracts only the addition of ATP to initiate the GS modification -reac- specifically required ATP, was inhibited by a-ketoglutarate, tion. a-Ketoglutarate inhibited the GS modification reaction and was stimulated by glutamine. Similarly, the adenylylation with both dialyzed and undialyzed extracts. The extract modi- reaction in E. coli is stimulated by glutamine and inhibited by fied in vitro along with the control extract was assayed for bio- a-ketoglutarate, and the ratio of these two metabolites regu- synthetic activity by using two common assays; the forward as- lates the adenylylation state of GS (4, 5). With E. coli, Stadtman say (13) and the radioactive assay (14). Both biosynthetic and coworkers have shown that .a complex of several enzymes activities as well as transferase activity were greatly decreased and proteins is required for the adenylylation/deadenylylation after modification of GS in crude extracts (Table 1). SVPDE reaction (4). treatment ofthe modified crude extracts restored GS activity to Adenylylation as a mechanism for regulating GS activity has the initial levels,, ruling out the possibility that the in vitro in- been examined by several groups in a variety of Gram-negative activation was due to an ATP-stimulated protease or any other and Gram-positive bacteria. Gancedo and Holtzer (6) concluded irreversible process. The requirement for.ATP, the stimulatory that adenylylation appeared to be present only in the enteric bacteria. Several Gram-positive bacteria as well as yeasts were Table 1. Effect ofin vitro modification on GS transferase and shown to lack GS modification upon addition ofammonium sul- biosynthetic activities fate to nitrogen-limited cultures, while in four strains ofenteric GS activity, U/mg bacteria GS activity was rapidly lost. Tronick et al. (7) also examined a number ofGram-positive and Gram-negative bacteria Biosynthetic for GS modification and came to conclusions similar to those of- Enzyme sample Transferase Forward Radioactive Gancedo and Holzer (6). Tronick et aL (7) based their conclu- Dialyzed crude sions on the effect of SVPDE on GS activity in extracts. All the extract 13.7 0.51 0.0600 Gram-negative bacteria examined showed a change in activity Adenylylated crude after SVPDE treatment, whereas the Gram-positive bacteria extract 0.23 0.02 0.0005 did not. The GS activity in B. polymyxa was altered by SVPDE treatment and may be adenylylated. However, this bacterium A crude extract of cells grown on glucose/glutamate medium was is as Tronick et al. also ex- prepared and a portion was adenylylated for 45 min under-standard classified being Gram-variable. (7) conditions. GS activity was determined by using three assays. Trans- amined two strains of Streptomyces, S. rutgersensis and S. dia- ferase and forward biosynthetic activity: 1 U = 1 gmol of Y-glutamyl- statochromogenes, and reported no indication ofadenylylation. hydroxamate formed per min. Radioactive biosynthetic assay: 1 U = When S. cattleya cells were grown under similar conditions GS 1 gmol of glutamine formed per min. was present in a very low adenylylation state and consequently Downloaded by guest on October 1, 2021 Biochemistry: Streicher and Tyler Proc. NatL Acad. Sci. USA 78 (1981) 233 activity was not altered by SVPDE treatment. Tronick et al. (7) 1. Prusiner, S. & Stadtman, E. R., eds. (1973) The Enzymes of Glu- also examined the immunological similarities of GS from the tamine Metabolism (Academic, New York), p. 615. various strains and found that all GS subject to adenylylation 2. Weglenski, P. & Tyler, B. (1977)1 Bcdteriol 129, 880-887. 3. Tyler, B. (1978) Annu. Rev. Biochem. 47, 1127-1162. crossreactedwith antisera to E. coli GS. A notable exception was 4. Ginsburg, A. & Stadtman, E. R. (1973) in The Enzymes of Gluta- the crossreaction found with the GS from the two Streptomyces mine Metabolism, eds. Prusiner, S. & Stadtman, E. R. (Academic, strains. However, in consideration of our results, and the pos- New York), pp. 9-43. sibility, discussed by Tronick et al. (7), that the crossreactivity of 5. Wohlhueter, R. R., Schutt, H. & Holzer, H. (1973) in The En- all the GS was due to the antigenicity ofthe adenylylation site, zymes of Glutamine Metabolism, eds. -Prusiner, S. & Stadtman, it is likely that the GSs ofthe other two Streptomyces strains are E. R. (Academic, New York), pp. 45-64. also modified by adenylylation. 6. Gancedo, C. & Holzer, H. (1968) Eur. J. Biochem. 4, 190-192. S. cattleyaCGS is also similar to the E. coli enzyme with respect 7. Tronick, S. R., Ciardi, J. E. & Stadtman, E. R. (1973)J. Bacteriol to the conditions necessary for-the transferase and the two bio- 115, 858-868. 8. Deuel, T. F., Ginsburg, A., Yeh, J., Shelton, E. & Stadtman, E. synthetic reactions, except for one striking difference. We R. (1970) J. Biol Chem. 245, 5195-5205. found that transferase activity, like the biosynthetic activities, 9. Hachimori, A., Matsunaga, A., Shimizu, M., Samejima, T. & No- declined upon adenylylation and that fully adenylylated GS was soh, Y. (1974). Biochim. Biophys. Acta 350, 461-474. almost inactive. Transferase activity was not restored by chang- 10. Wedler, F. C. & Hoffmann, F. M. (1974) Biochemistry 13, ing either Mn21 or Mg2+ concentration or pH, and thus we can- 3207-3214. not determine the total amount ofGS in an extract orwhole cells 11. Kahan, J. S., Kahan, F. M., Goegelman, R., Currie, S. A., Jack- by means of a standard assay. The total amount ofGS in extracts son, M., Stapley, E. O., Miller, T. W., Hendlin, D., Mochales, can be determined by treating a sample with SVPDE and assay- S., Hernandez, S., Woodruff, H. B. & Birnbaum, J. (1979)J. An- ing for transferase activity after complete digestion. Comparing tibiot. 32, 1-12. the transferase activity before and after SVPDE incubation, we 12. Streicher, S. L. & Tyler, B. (1980) Fed. Proc.-Fed. Am. Soc. Exp. can estimate the adenylylation state. This also can be done in- Biol 39, 1775 (abstr.). dependent of enzyme assays by analysis of extracts on two-di- 13. Bender, R. A., Janssen, K. A., Resnick, A. D., Blumenberg, M., mensional gels in which the adenylylated and unadenylylated Foor, F. & Magasanik, B. (1977)J. Bacteriol 129, 1001-1009. Subunits are clearly separated. 14. Tiemeier, D. C. & Milman, G. (1972) J. Biol Chem. 247, We have not addressed in this paper the regulation ofsynthe- 2272-2277. sis of GS in Streptomyces. There appears to be significant con- 15. Foor, F., Janssen, K. A. & Magasanik, B. (1975) Proc. Natl Acad. trol ofthe levels ofGS protein in cells in response to the source Sci. USA 72, 4844-4848. of nitrogen in the growth medium (21). Comparing data from 16. O'Farrell, P. H. (1975)J. Biol Chem. 250, 41)07-4021. enzyme assays and two-dimensional gel analysis, we find more 17. Bender, R. A. & Streicher, S. L. (1979) J. Bacteriol 137, than a 20-fold difference in GS protein levels between re- 1000-1007. 18. Ludwig, R. A. (1980)J. Bacteriol 141, 1209-1216. pressed and derepressed cultures. At this time we cannot attrib- 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. ute this level ofcontrol to changes in the rate oftranscription of (1951)3. Biol Chem. 193, 265-275. the S. cattleya ginA gene or to enhanced or decreased rates of 20. Sims, A. P., Toone, J. & Box, V. J. (1974) 1. Gen. Microbiol 84, degradation ofeither the glnA mRNA or the GS subunit. 149-162. We thank Gary Roberts for preparation of the two-dimensional gels, 21. Aharonowitz, Y. (1979) in Genetics ofIndustrial Microorganisms, Philip Paress for providing purified GS, and Forrest Foor for stimu- eds. Sebek, 0. K. & Laskin, A. I. (Am. Soc. Microbiol., Washing- lating discussions and encouragement. ton, DC), pp. 210-217. Downloaded by guest on October 1, 2021