Proc. NatL Acad. Sci. USAVol. 78, No. 1, pp. 229-233, January 1981Biochemistry

Regulation ofglutamine synthetase activity by adenylylation in theGram-positive bacterium Streptomyces cattleya

(nitrogen metabolism/covalent modification of proteins)

STANLEY L. STREICHER AND BONNIE TYLERMerck 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[GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]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-cline in GS activity. Crude extracts prepared from such ammonia-shocked cells had very low levels ofGS activity as measured by bio-synthetic and y-glutamyltransferase assays. Incubation of thecrude extracts with snake venom phosphodiesterase restored GSactivity. In cell extracts, GS was also inactivated by an ATP- andglutamine-dependent reaction. Radioactive labeling studies dem-onstrated the incorporation of an AMP moiety into GS proteinupon modification. Our results suggest a covalent modification ofGS in a Gram-positive bacterium. This modification appears to beadenylylation of the GS subunit similar to that found in the Gram-negative bacteria.

Glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] is responsible for the synthesis of gluta-mine from glutamic acid and ammonia:

glutamate + NH3 + ATP -- glutamine + ADP + Pi.The enzyme occupies a central position in nitrogen metabolismbecause the amide nitrogen ofglutamine is used for the synthe-sis ofmany metabolites (1). In the enteric bacteria such as Esch-erichia coli, the ability ofthe cell to synthesize glutamine is reg-ulated at two levels. The amount of GS protein in the cell isregulated at the level of transcription of the glnA gene (2). Therate of glnA transcription is inversely proportional to the avail-ability ofnitrogen (3). The ability ofthe GS protein to synthesizeglutamine is controlled through covalent modification. The ad-dition and removal ofan AMP moiety (adenylylation/deadenyl-ylation) alters the biosynthetic activity of the enzyme (4, 5).High levels of adenylylation and low biosynthetic activity arenormally found when nitrogen is in excess and the converse isfound when nitrogen is limiting (3-5). In several other Gram-negative bacteria GS activity is also regulated through adenylyl-ation (3, 6, 7). This is in contrast to the situation in the Gram-positive bacilli. In both Bacillus subtilis (8) and B. stearother-mophilis (9, 10), from which GS has been purified and exam-ined, no evidence was obtained to suggest covalent modifica-tion.We have been investigating nitrogen metabolism in the

Gram-positive filamentous spore-forming bacterium Strepto-myces cattleya (11) and have initially examined the GS in thisorganism. We describe here results demonstrating that the ac-tivity ofGS in S. cattleya is regulated through a covalent modi-fication that appears to be similar to that found in the entericbacteria. We believe that adenylylation ofGS in a Gram-positive

bacterium has not been reported previously. [A preliminary re-port of this work has been presented (12). ]

MATERIALS AND METHODS

Bacterial Strain and Culture Conditions. For all experi-ments the original soil isolate of S. cattleya was used (11). Cellswere grown in a mineral salts medium (D medium) supple-mented with 1% glucose and 20 mM sodium glutamate. D me-dium contains, per liter; 0.3 g ofK2HPO4, 0.5 g ofNaCl, 0.5 g ofMgSO4-7H2O, 19.5 g of 2-(N-morpholino)ethansulfonic acid(Mes) at pH 7, 10 mg of CoCl2, 25 mg of FeSO4-7H2O, and 10mg ofZnSO4-7H20. Spores were inoculated into the medium ata concentration of 10' per ml and incubated at 370C with shakingfor 24-36 hr. Cells were harvested by centrifugation or filtrationand stored at -800C.

Preparation of Crude Extracts. Frozen cells were resus-pended in cold buffer A (20 mM imidazole-HCl, pH 7.5/1 mMMnCl2) at a concentration of0. 2-0.5 g ofcells per ml. Cells weredisrupted by intermittent sonic oscillation for a total time of 1min. Debris was removed by centrifugation for 30 min at20,000 X g. Extracts were stored at either 00C or -80'C. Fro-zen extracts maintained GS activity for several months.GS Assays. The y-glutamyltransferase assay was performed

essentially as described by Bender et al. (13) atpH 6.9. The "for-ward" biosynthetic assay, which measures the formation of y-glutamylhydroxamate from glutamate, hydroxylamine, and ATPwas performed at pH 7.3 as described (13). The "radioactive"biosynthetic assay measuring the formation of [3H]glutaminefrom [3H]glutamate, NH3, and ATP was performed at pH 7.3 asdescribed (14). One enzyme unit (U) equals 1 ,umol of y-gluta-mylhydroxamate or [3H]glutamine formed per min.

Adenylylation Reaction. Modification ofGS in crude extractswas performed as described by Foor et al. (15). Where indicatedthe concentrations of ATP and glutamine were altered. Reac-tions were started by addition ofcrude extract to prewarmed re-action mixture. At indicated times samples were removed andadded to chilled GS assay mixtures.

Snake Venom Phosphodiesterase (SVPDE) Treatment. Ex-tracts were incubated at 37°C with SVPDE (Boehringer Man-nheim) at 100 ,ug/ml and samples were removed for analysis asdescribed in the text.

Radioactive Labeling Studies. Cells were labeled with inor-ganic [32P]phosphate in minimal medium modified to contain 1/10th the usual concentration of phosphate. When the cell den-sity reached 100 Klett units (no. 54 filter), 0.25 mCi (1 Ci = 3.7x 10'0 becquerels) of 32pi was added to 100 ml of culture. After15 min, 50 ml of the culture was harvested and the remainder

Abbreviations: GS, glutamine synthetase; SVPDE, snake venom phos-phodiesterase; U, enzyme unit.

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230 Biochemistry: Streicher and Tyler

received ammonium chloride (20 mM). After an additional 15min these cells were harvested. GS present in crude extractswas labeled by performing the standard adenylylation reactionin the presence of either [a-32P]ATP or [adenine-3H]ATP (0.25mCi per 3-ml reaction mixture) for 30 min.

Gel Electrophoresis. Two-dimensional gel electrophoresiswas carried out by using the procedures of O'Farrell (16). Pro-teins were visualized with either Coomassie blue staining or au-toradiography, using standard procedures. Fluorography wasdone with En3Hance (New England Nuclear). NaDodSO4 gelswere run by using the procedure of Bender and Streicher (17),and nondenaturing gels by the method of Ludwig (18).

Protein Determination. Protein concentration was deter-mined by the procedure of Lowry et al. (19) or by the Bio-Raddye binding method (according to the supplier's instructions).

Chemicals. All reagents were ofthe highest quality commer-cially available. Radioactive compounds were obtained fromNew England Nuclear.

RESULTSModification of S. cattleya GS in Whole Cells. Crude ex-

tracts of S. cattleya grown in a minimal salts medium containing

60

0-)

600

, 50

40

300-

20

10-~~~~~~~~

10

-5 0 5 10 15

Time (min)

FIG. 1. Kinetics of GS inactivation in whole cells. S. cattleya cellsgrowing in glucose/glutamate minimal medium were assayed for GStransferase activity by taking 80-pl samples and adding them to coldassay mixture containing cetyltrimethylammonium bromide at 100gg/ml. At 0 min ammonium chloride was added (to 20 mM) and sam-pling was continued. After all samples were taken transferase assayswere run at 370C for 20 min.

*100

80

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0

0-

60

50

40

30

20

)-. 8 0

o-~~0

6oxL1 0

0n - - - .S -0/00' ~ M

100o- ... - _

0o I I0 10 20 30 40 50 60 70 80 90

Incubation Time (min)

FIG. 2. Effect of SVPDE on GS transferase activity in crude ex-tracts ofammonia-shocked and unshocked cells. A culture ofS. cattleyacells growing in glucose/glutamate minimal medium was split into twoparts. One part was harvested while the other received ammoniumchloride (to 20 mM). After a 30-min "ammonia shock" period these cellswere harvested. Crude extracts were prepared and samples weretreated with SVPDE. At the indicated time points 5-Al samples wereremoved and added to cold transferase assay mixture. Samples wereassayed at 37"C for 10 min. Ammonia-shocked extract: m, no SVPDE;n, with SVPDE. Unshocked control extract: 0, no SVPDE; o, withSVPDE.

glucose and glutamate as the sources of carbon and nitrogenhave high levels ofGS as determined by the y-glutamyltransfer-ase assay (about 7-10' U/mg of protein). Upon the addition ofammonium chloride to a mid-logarithmic culture we observeda rapid decline in whole cell transferase activity (Fig. 1) to a levelabout 1/7th that of the initial one. Crude extracts of these am-monia-shocked cells also had a lower level oftransferase activitycompared to the control extracts (about 1 U/mg of protein) andhad a much lower level of forward biosynthetic activity. Theseresults suggested that modification of GS occurred in responseto 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 byadenylylation (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 inactivatesGS by causing the conversion of native octameric GS into lessactive tetramers and then into inactive monomers (20).We initially characterized the modification of S. cattleya GS

by examining the pH profile and divalent metal requirements ofextracts from active and ammonia-shocked cells. Although theamount of transferase activity was low in extracts of ammonia-shocked cells, the pH profile was essentially the same as that ofthe control extract. * Similarly there was no effect of addingMg2e (60mM) to the transferase reaction with either extract. In-cubation of S. cattleya extracts with SVPDE, known to cleavetheAMP moiety ofadenylylated E. coli GS, had a significant ef-fect on the activity in the extract from ammonia-shocked cells(Fig. 2). GS transferase activity increased nearly to that of the

* Wax, R. & Snyder, L. (1980) Annual Meeting of the American Societyfor Microbiology, Miami Beach, FL, p. 187 (abstr.).

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Proc. Nati. Acad. Sci. USA 78 (1981) 231

A B

p..

C

aC D"~~~~~~..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 ofC (boxed area). Arrows point to

the nonadenylylated GS subunit (the more basic ofthe pair).

-control extract. There was no alteration ofactivity in the control..extract~treated with SVPDE or an increase in activity of the ex-tract of ammonia-shocked cells in the absence of SVPDE. Insimilar experiments we monitored forward biosynthetic activityand found that it also significantly increased after SVPDE treat-ment (data not shown). We conclude from these results that S.cattleya GS is modified after ammonia shock to an inactive formthrough a covalent linkage involving a phosphodiester bond.The addition ofa phosphodiester group to a protein would be

expected to alter its charge and therefore the isoelectric point.We analyzed extracts from control and ammonia-shocked cellsby using the O'Farrell two-dimensional polyacrylamide gelelectrophoresis technique (16) to separate proteins by isoelec-tric point as well as molecular weight. The only noticeable dif-ference in the protein profiles of the two extracts was found inthe amounts oftwo proteins differing only in charge (Fig. 3). Inthe control extract the more basic protein ofthe pair was presentin a much higher amount than the more acidic one. However inthe extract from ammonia-shocked cells the situation was re-versed: the more acidic protein was present in a much higheramount than the more basic one. After treatment of the latterextract with SVPDE the pattern reversed and the more acidicprotein was barely detectable -(gel not shown). The change inrelative amounts of these two proteins is consistent with theirbeing the modified and unmodified forms ofthe GS subunit. To

confirm this interpretation we purified S. cattleya GS andshowed that the protein pair in question represented the inter-convertible forms ofthe GS subunit (unpublished). We obtainedadditional evidence that the change in isoelectric point was dueto the phosphodiester linkage by incorporating inorganic[32P]phosphate into GS. In this experiment we added[32P]phosphate to a mid-logarithmic culture and harvested cellsbefore and after ammonia shock. Extracts were prepared andanalyzed on two-dimensional gels. When we compared thestained gels with the autoradiographs we observed that 32p wasincorporated only into the more acidic protein of the GS pair.The amount of 32p incorporated into the GS protein greatly in-creased after ammonia shock, and all the radioactivity in the GSprotein was removed after treatment with SVPDE. 32p was alsoincorporated into several other proteins. A few of these appearto contain phosphodiester bonds, because the radioactivity wasremoved by SVPDE incubation. We do not know the identity ofthese proteins.

Modification of S. cattleya GS in Crude Extracts. The addi-tion ofATP to crude extracts caused a rapid loss ofGS transferaseactivity (Fig. 4). ATP was specifically required for the modifi-cation reaction; UTP, CTP, and GTP were all inactive. We wereable to demonstrate a stimulatory effect ofglutamine on the rateof modification (Fig. 4B) when we used dialyzed extracts.Crude extracts prepared by our standard procedures required

Biochemistry: Streicher and Tyler

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4, W,

.-J

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232 Biochemistry: Streicher and Tyler

70

.~60 60-

<

50 m50

o~~ ~ ~~~~Tminn)

FI 40 -Pand u 40n oA

30 30 V20 %20-

0 ~~~~~U

0 jZR I

0 510O1520'25 30 0 510 152025 30

Time (min)

FIG. 4. ATP and glutamine dependence ofCS inactivation in crude

extracta. A crude, extract was prepared from cells grown on glucose/

glutamate medium and was -dialyzed overnight against 500 vol of

buffer A. Then 0.2 ml ofcrude extractwas added to 0.2 ml ofprewarmedadenylylation reaction mixture (15). At the indicated times-5-,ul sam-

ples were taken, added to cold transferase reaction mixtures, and, atthe completion of the sampling, assayed at 370C for 10 min. (A) Effectofvarying the ATP concentration. ATP concentrations were: o,0 mM;*, 1 mM; *, 2 mM; *, 3 mM; A, 5 mM; n, 10 mM. (B) Effect ofvaryingthe glutamine concentration. Glutamine concentrations were: e, 0

mM; A, 2.5 mM; m,5- mM; o, 10mM; o, 30mM.

only the addition of ATP to initiate the GS modification -reac-

tion. a-Ketoglutarate inhibited the GS modification reactionwith both dialyzed and undialyzed extracts. The extract modi-fied in vitro along with the control extract was assayed for bio-synthetic activity by using two common assays; the forward as-

say (13) and the radioactive assay (14). Both biosyntheticactivities as well as transferase activity were greatly decreasedafter modification of GS in crude extracts (Table 1). SVPDEtreatment of the modified crude extracts restored GS activity tothe initial levels,, ruling out the possibility that the in vitro in-activation was due to an ATP-stimulated protease or any otherirreversible process. The requirement for.ATP, the stimulatory

Table 1. Effect of in vitro modification on GS transferase andbiosynthetic activities

GS activity, U/mgBiosynthetic

Enzyme sample Transferase Forward Radioactive

Dialyzed crudeextract 13.7 0.51 0.0600

Adenylylated crudeextract 0.23 0.02 0.0005

A crude extract of cells grown on glucose/glutamate medium wasprepared and a portion was adenylylated for 45 min under-standardconditions. GS activity was determined by using three assays. Trans-ferase and forward biosynthetic activity: 1 U = 1 gmol of Y-glutamyl-hydroxamate formed per min. Radioactive biosynthetic assay: 1 U =1 gmol of glutamine formed per min.

effect of glutamine, and the inhibitory effect of a-ketoglutarateon GS modification are very similar to the conditions for adenyl-ylation of E. coli GS. We modified the GS in crude extracts inthe presence of either [a-32P]ATP or [adenine-3H]ATP. The in-activated GSs present in both extracts were analyzed by usingnondenaturing 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-denaturing gel. In this case we also found that modification ofGS did not result in a significant change in the native enzymestructure. The position of the labeled GS coincided with thestained GS and had mobility identical to that of purified activeGS. Therefore it is clear that adenylylation of GS, while causinga drastic loss ofenzymatic activity, did not also cause aggregationor disassembly of the native enzyme. These labeling studiesprovide strong evidence that S. cattleya GS is modified byadenylylation.

DISCUSSION

S. cattleya is a Gram-positive filamentous spore-forming bacte-rium (11). In most respects it is quite different from the entericbacteria. We have shown here, however, that with respect toglutamine synthetase there is a remarkable similarity. In a pre-liminary report, Wax and Snyder* found that an ammonia shockof S. cattleya cells growing on lysine as the source ofnitrogen ledto a rapid loss ofGS transferase activity. Our experiments dem-onstrate that when S. cattleya cells growing under nitrogen-lim-iting conditions were exposed to ammonium chloride a rapidmodification ofGS occurred, with the concomitant loss ofall GSactivities. We found that full activity was recovered by treat-ment of crude extracts with SVPDE. This suggested that themodification of GS involved a phosphodiester linkage. Radiola-beling experiments also established that the adenine moiety ofATP was covalently incorporated into GS subunits, leading us toconclude that the enzymatic activity of S. cattleya GS is 'regu-lated by adenylylation. Adenylylation of GS in crude extractsspecifically required ATP, was inhibited by a-ketoglutarate,and was stimulated by glutamine. Similarly, the adenylylationreaction in E. coli is stimulated by glutamine and inhibited bya-ketoglutarate, and the ratio of these two metabolites regu-lates the adenylylation state of GS (4, 5). With E. coli, Stadtmanand coworkers have shown that .a complex of several enzymesand proteins is required for the adenylylation/deadenylylationreaction (4).

Adenylylation as a mechanism for regulating GS activity hasbeen examined by several groups in a variety of Gram-negativeand Gram-positive bacteria. Gancedo and Holtzer (6) concludedthat adenylylation appeared to be present only in the entericbacteria. Several Gram-positive bacteria as well as yeasts wereshown to lack GS modification upon addition ofammonium sul-fate to nitrogen-limited cultures, while in four strains of entericbacteria GS activity was rapidly lost. Tronick et al. (7) also ex-amined a number of Gram-positive and Gram-negative bacteriafor GS modification and came to conclusions similar to those of-Gancedo and Holzer (6). Tronick et aL (7) based their conclu-sions on the effect of SVPDE on GS activity in extracts. All theGram-negative bacteria examined showed a change in activityafter SVPDE treatment, whereas the Gram-positive bacteriadid not. The GS activity in B. polymyxa was altered by SVPDEtreatment and may be adenylylated. However, this bacteriumis classified as being Gram-variable. Tronick et al. (7) also ex-amined two strains of Streptomyces, S. rutgersensis and S. dia-statochromogenes, and reported no indication of adenylylation.When S. cattleya cells were grown under similar conditions GSwas present in a very low adenylylation state and consequently

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Proc. NatL Acad. Sci. USA 78 (1981) 233

activity was not altered by SVPDE treatment. Tronick et al. (7)also examined the immunological similarities of GS from thevarious strains and found that all GS subject to adenylylationcrossreacted with antisera to E. coli GS. A notable exception wasthe crossreaction found with the GS from the two Streptomycesstrains. However, in consideration of our results, and the pos-sibility, discussed by Tronick et al. (7), that the crossreactivity ofall the GS was due to the antigenicity of the adenylylation site,it is likely that the GSs ofthe other two Streptomyces strains arealso modified by adenylylation.

S. cattleyaCGS is also similar to the E. coli enzyme with respectto the conditions necessary for-the transferase and the two bio-synthetic reactions, except for one striking difference. Wefound that transferase activity, like the biosynthetic activities,declined upon adenylylation and that fully adenylylated GS wasalmost inactive. Transferase activity was not restored by chang-ing either Mn21 or Mg2+ concentration or pH, and thus we can-not determine the total amount ofGS in an extract or whole cellsby means ofa standard assay. The total amount ofGS in extractscan be determined by treating a sample with SVPDE and assay-ing for transferase activity after complete digestion. Comparingthe transferase activity before and after SVPDE incubation, wecan estimate the adenylylation state. This also can be done in-dependent of enzyme assays by analysis of extracts on two-di-mensional gels in which the adenylylated and unadenylylatedSubunits are clearly separated.We have not addressed in this paper the regulation ofsynthe-

sis of GS in Streptomyces. There appears to be significant con-trol ofthe levels ofGS protein in cells in response to the sourceof nitrogen in the growth medium (21). Comparing data fromenzyme assays and two-dimensional gel analysis, we find morethan a 20-fold difference in GS protein levels between re-pressed and derepressed cultures. At this time we cannot attrib-ute this level ofcontrol to changes in the rate of transcription ofthe S. cattleya ginA gene or to enhanced or decreased rates ofdegradation ofeither the glnA mRNA or the GS subunit.We thank Gary Roberts for preparation of the two-dimensional gels,

Philip Paress for providing purified GS, and Forrest Foor for stimu-lating discussions and encouragement.

1. Prusiner, S. & Stadtman, E. R., eds. (1973) The Enzymes of Glu-tamine Metabolism (Academic, New York), p. 615.

2. Weglenski, P. & Tyler, B. (1977)1 Bcdteriol 129, 880-887.3. Tyler, B. (1978) Annu. Rev. Biochem. 47, 1127-1162.4. Ginsburg, A. & Stadtman, E. R. (1973) in The Enzymes of Gluta-

mine Metabolism, eds. Prusiner, S. & Stadtman, E. R. (Academic,New York), pp. 9-43.

5. Wohlhueter, R. R., Schutt, H. & Holzer, H. (1973) in The En-zymes of Glutamine Metabolism, eds. -Prusiner, S. & Stadtman,E. R. (Academic, New York), pp. 45-64.

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3207-3214.11. Kahan, J. S., Kahan, F. M., Goegelman, R., Currie, S. A., Jack-

son, M., Stapley, E. O., Miller, T. W., Hendlin, D., Mochales,S., Hernandez, S., Woodruff, H. B. & Birnbaum, J. (1979)J. An-tibiot. 32, 1-12.

12. Streicher, S. L. & Tyler, B. (1980) Fed. Proc. -Fed. Am. Soc. Exp.Biol 39, 1775 (abstr.).

13. Bender, R. A., Janssen, K. A., Resnick, A. D., Blumenberg, M.,Foor, F. & Magasanik, B. (1977)J. Bacteriol 129, 1001-1009.

14. Tiemeier, D. C. & Milman, G. (1972) J. Biol Chem. 247,2272-2277.

15. Foor, F., Janssen, K. A. & Magasanik, B. (1975) Proc. Natl Acad.Sci. USA 72, 4844-4848.

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1000-1007.18. Ludwig, R. A. (1980)J. Bacteriol 141, 1209-1216.19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

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