In-vivo modification of Azotobacter-chroococcum glutamine-synthetase

In i o modi ica ion o Azo obac e ch oococcum glu amine syn he ase Ma ia C. MUNOZ-CENTENO, F ancisco J. CEJUDO and An onio PANEQUE* Ins i u o de Bioqu mica Vege al y Fo os n esis, Uni e sidad de Se illa-Consejo Supe io de In es igaciones Cien icas, Apa ado 1113, 41080 Se illa, Spain A monospeci ic an i-(glu amine syn he ase) an ibody aised agains glu amine syn he ase o he unicellula cyanobac e ium Synechocys is sp. s ain PCC 6803 immuno eac ed wi h glu amine syn he ase om he N2- ixing he e o ophic bac e ium Azo obac e ch oococcum. In Wes e n-blo ing expe imen s his an ibody ecognized a single p o ein o a molecula mass o 59 kDa co esponding o glu amine syn he ase subuni . This p o ein was in i o-labelled in esponse o addi ion o ammonium, bo h [3H]adenine and H332PO4 p eincuba ion o he cells being equally e ec i e. Ne e heless, he amoun o glu amine INTRODUCTION The azo obac e s a e he e o ophic ni ogen- ixing ae obic bac- e ia ha can use, besides molecula ni ogen, o he ino ganic ni ogen sou ces such as ni a e o ni i e. Ammonium, he p oduc o he educ ion o N2 and ni a e, con ols in i s u n he ac i i ies o ni ogenase [1-3] and ni a e up ake [4], bo h ene gy- dependen e en s. In Azo obac e ch oococcum [5] and A. inelandii [6] ammonium is inco po a ed in o ca bon skele ons ia he glu amine syn he ase (GS)-glu ama e syn hase (GOGAT) ou e, which is hough o be solely esponsible o he as- simila ion o ammonium. Since GS, ha ca alyses an ATP- equi ing eac ion, plays a pi o al ole in ni ogen me abolism in many p oka yo es and euka yo es, bo h he syn hesis and ac i i y o his enzyme ha e been conside ed o be s ic ly egula ed in esponse o he a ailable ni ogen sou ce. Regula ion o GS by gene exp ession, eedback inhibi ion, and co alen modi ica ion has been s udied ex ensi ely in En e o- bac e ia [7-9]. In Esche ichia coli, GS is a dodecame o 12 iden ical subuni s, each o which can be egula ed independen ly by e e sible adenylyla ion ende ing a less-ac i e enzyme. The adenylyla ion-deadenylyla ion eac ions a e ca alysed by adenylyl ans e ase, he ac i i y o which is egula ed by he egula o y p o ein PII which i sel is egula ed h ough e e sible u idylyla ion ca ied ou by an u idylyl ans e ase. The la e enzyme esponds o he a io o glu amine o a-oxoglu a a e, shi ing GS o he mo e adenylyla ed s a e as he a io o glu amine o a-oxoglu a a e inc eases and ice e sa [10,1 1]. The adenylyla ion-deadenylyla ion sys em is ope a i e in mos G am- nega i e [12-15] bu no in g am-posi i e [16] bac e ia, he me hanogen Me hanobac e ium i ano i [17] o cyanobac e ia [18,19]. Besides his co alen modi ica ion, GS om bo h p oka yo ic and euka yo ic o ganisms has been desc ibed o be ADP- ibosyla ed in i o, and in some cases he eac ion was accompanied by he loss o GS ac i i y ([13] and e e ences ci ed he ein). The in i o exis ence o his modi ica ion has no ye been epo ed, howe e . In o ma ion on azo obac e GS is e y sca ce. The enzyme has been pu i ied om A. inelandii and shown o con ain AMP syn he ase p esen in A. ch oococcum was independen o he a ailable ni ogen sou ce. Modi ied, inac i e glu amine syn he ase was e-ac i a ed by ea men wi h snake- enom phosphodies e ase bu no by alkaline phospha ase. L- Me hionine-DL-sulphoximine, an inhibi o o glu amine syn he ase, p e en ed he enzyme om being co alen ly modi ied. We conclude ha , in A. ch oococcum, glu amine syn he ase is adenylyla ed in esponse o ammonium and ha o he modi ica ion o ake place ammonium mus be me abolized. bound co alen ly when isola ed om ammonium-g own cells [20,21]. Recen ly i has been desc ibed ha , in A. inelandii, exp ession o glnA, he gene encoding glu amine syn he ase, is no con olled by he a ailable ni ogen sou ce [22]. He e we epo ha GS o A. ch oococcum did c oss- eac wi h an ise um aised agains GS om he cyanobac e ium Synechocys is sp. PCC 6803. We also show ha , in A. ch oococcum, GS is egula ed by adenylyla ion-deadenylyla ion in esponse o he addi ion o ammonium o he g ow h medium, wi h ammonium de e mining he GS ac i i y le el a he han he enzyme syn hesis. Finally, we epo ha in he p esence o L-me hionine-DL-sulphoximine (MSX), an inhibi o o GS, adenylyla ion o his enzyme was p e en ed. MATERIALS AND METHODS Ma e ials Coomassie B illian Blue R-250 was om Bio-Rad, Richmond, CA, U.S.A. Alkaline phospha ase and phosphodies e ase we e om Boeh inge -Mannheim, Mannheim, Ge many. [3H]Adenine (25.8 Ci/mol) and H332PO4 (8800 Ci/mmol) we e pu chased om du Pon -New England Nuclea , D eieich, Ge many. Fo de- e mina ion o molecula mass, an elec opho esis calib a ion ki om Bio-Rad Labo a o ies, He cules, CA, U.S.A., was used. All o he chemicals we e o analy ical g ade. O ganism and g ow h condi ions A. ch oococcum A.T.C.C. 4412 ( om he Uni e si y o Valencia Collec ion, Valencia, Spain) was g own on ni ogen- ee Bu k's medium supplemen ed wi h 0.5 % (w/ ) suc ose as he sole ene gy and ca bon sou ce. When indica ed, his medium was supplemen ed wi h NH4C1 o KNO3 a he concen a ion speci ied in each case. G ow h condi ions we e as p e iously desc ibed [23]. Cell ex ac s Cells we e ha es ed by cen i uga ion a 10000 g o 10 min a 4 °C, washed in 50 mM Mops/KOH bu X, pH 7.5, and Abb e ia ions used: GS, glu amine syn he ase; GOGAT, glu ama e syn hase; MSX, L-me hionine-DL-sulphoximine. * To whom co espondence should be add essed. 641 Biochem. J. (1994) 298, 641-645 (P in ed in G ea B i ain) 642 M. C. Munoz-Cen eno, F. J. Cejudo and A. Paneque esuspended a a concen a ion o 1 g o cells/3 ml o 50 mM Mops/KOH bu e , pH 7.5, con aining 1 mM DL-di hio h ei ol and 1 mM phenylme hanesulphonyl luo ide. Cells we e dis up ed by sonica ion (20 kHz; 75 W) o 5 min (in 30 s pe iods) wi h a B anson soni ie model B 12. The homogena e was cen i uged a 33000 g o 30 min a 4 °C, and he esul ing supe na an cons i u ed he cell ex ac . 20 min and he supe na an cons i u ed he labelled cell ex ac . Whe e indica ed, immunop ecipi a ion o labelled p o eins was ca ied ou as desc ibed abo e o he unlabelled cell ex ac . When gels we e loaded wi h p o eins om cell ex ac s ha we e no immunop ecipi a ed, he GS p o ein was iden i ied by Wes e n-blo (immunoblo ing) analysis. Analy ical me hods Enzyme assays GS ac i i y was de e mined in i o by he o ma ion o y- glu amylhyd oxama e ( ans e ase assay) in he p esence o ei he Mn2+ (inac i e o m) o Mn2+ plus Mg2+ (ac i e GS o m) [21,24]. Ni a e educ ase ac i i y was de e mined in i o wi h di hioni e- educed Me hyl Viologen as he elec on dono [25]. lmmunop ecipi a ion o GS p o ein The A. ch oococcum GS p o ein was immunop ecipi a ed om cell ex ac s wi h an ise um di ec ed agains he Synechocys is sp. s ain PCC 6803 GS (kindly p o ided by F. J. Flo encio, om his Ins i u e) as ollows: 0.3 ml aliquo s o ex ac s om A. ch oococcum cells we e mixed wi h inc easing amoun s o he c ude abbi (5 mg o p o ein/ml) an ise um. The immuno- p ecipi a e was allowed o o m o e nigh a 4 °C and hen cen i uged a 10000 g o 10 min a 4 'C. The pelle was dissol ed in 40 ,ul o sample bu e [26] o SDS/PAGE, and he supe na an sa ed o de e mining he emaining ac i i ies o GS and, whe e indica ed, ni a e educ ase. Wes e n blo (immunoblo ing) A e SDS/PAGE, he p o eins we e ans e ed o ni ocellulose shee s as in [27], using a mini-T ans Blo elec opho e ic ans e cell (Bio-Rad). To immunode ec p o eins, he ni ocellulose il e s we e blocked o e nigh in 200 mM NaCl and 15 mM T is/HCl, pH 7.4 (T is/NaCl), con aining 0.2 % (w/ ) sodium azide and 50% (w/ ) d ied skimmed milk. An i-GS an ibody (1:500) was added, and he mix u e incuba ed wi h shaking o e nigh . The il e s we e washed ou imes wi h T is/NaCl con aining 0.05 % ( / ) Tween 20, hen pe oxidase-conjuga ed an i-( abbi IgG) se um (Sigma) was used as second an ibody, and blo s we e de eloped as desc ibed by Komb ink e al. [28]. In i o [3H]adenine-labelling (32P-labelling) expe imen s A 2 ml cul u e was g own o mid-loga i hmic phase [a enuance (D560) o app ox. 0.5] in ni ogen- ee medium. Cells we e ha es ed by cen i uga ion a 7000 g o 5 min a oom em- pe a u e and esuspended in ml o cul u e medium bu e ed wi h 50 mM Mops/KOH, pH 7.5, and wi hou any po assium phospha e. Cells we e incuba ed a 30 'C wi h con inuous shaking (100 s okes min-') o 10 min and hen supplemen ed wi h ei he 19 ,uCi o [3H]adenine o 2 ,uCi o [32P]o ho- phospho ic acid. A e u he incuba ion o 2 h, 5 mM NH4C1 was added, and he cells we e ha es ed h la e . Sedimen a ion was imp o ed by adding NaCl and EDTA o inal concen a ions o 100 mM and l0 mM espec i ely. The cells we e esuspended in 40 ,u1 o sample bu e [26] o elec opho esis and dis up ed by eezing in liquid ai ollowed by boiling o 3 min ( epea ed ou imes a leas ). The homogena e was cen i uged a 16500 g o P o ein in cell ex ac s was es ima ed by he me hod o Ma kwell e al. [29] using BSA as s anda d. SDS/PAGE was pe o med acco ding o Laemmli [26]. P o ein ma ke s we e elec opho esed in pa allel. The p o eins we e s ained wi h Coomassie B illian Blue R-250. Fluo og ams o in i o-labelled p o eins we e ob ained as desc ibed in [30]. RESULTS An ibodies aised agains GS om Synechocys is c oss- eac wi h A. ch oococcum GS Addi ion o inc easing amoun s o an i-GS an ibody aised agains he enzyme om he cyanobac e ium Synechocys is o an A. ch oococcum cell- ee ex ac esul ed in p og essi e dis- appea ance o GS ac i i y (Figu e 1). Though no shown, i could be asce ained ha he an ise um was equally e ec i e in he immunop ecipi a ion o bo h ac i e and inac i e GS o m. As illus a ed in Figu e 1, no e ec o he an ibody was obse ed on ni a e educ ase ac i i y, which was used as a nega i e con ol. Fu he mo e, he analysis o he immunop ecipi a ed ma e ial on SDS/PAGE e ealed en ichmen o a p o ein o app ox. 59 kDa o molecula mass (Figu e 2) ha was no p esen in he an ise um alone (lane 7 in Figu e 2). To ou knowledge, he molecula mass o A. ch oococcum GS has no been epo ed p e iously. The A. inelandii GS, howe e , has been desc ibed as a dodecame wi h monome o molecula mass o ei he 56.5 [21], 53 [20] o 62 kDa, in he la e case when exp essed in Esche ichia coli [22]. On he basis o hese da a we concluded ha he p o ein en iched in he immunop ecipi a e co esponded o he GS monome . Taking in o conside a ion ha GS could be de ec ed on SDS/PAGE, we nex in es iga ed whe he o no his enzyme g 110 100io 0 90 Co(U CD 80 70 2n 60 ~ 50 40 o 30 0 -10 20 30 40 50 An i-GS (#l) Figu e 1 Immuno i a ion o A. ch oococcum GS Aliquo s (300 ,ul) o cell ex ac (3 mg p o ein/ml) om 8 mM KNO3-g own A. ch oococcum cells we e incuba ed wi h inc easing olumes o he an ibodies (5 mg/ml) aised agains pu e GS om Synechococcus sp. s ain PCC 6301. The ac i i ies o glu amine syn he ase (@) and ni a e educ ase (0) emaining in supe na an s we e measu ed as desc ibed in he Ma e ials and me hods sec ion. The le el o 100% GS ac i i y co esponds o 250 muni s/mg o p o ein; 100% ni a e educ ase ac i i y o he con ol was 20 muni s/mg o p o ein. Azo obac e ch oococcum glu amine syn he ase 643 M (kDa) 1 2 3 4 5 6 7 97.4 - 66.2 - - GS 40 "_ < IgG 45 .. . .! - *M .-.-.. .-.:U _.s. 31 - Figu e 2 E ec o an l-GS an ibody concen a ions on immunop ecipl a ion o A. ch oococcum GS Samples desc ibed in Figu e 1 we e cen i uged and he co esponding immunop ecipi a ed p o eins we e elec opho esed in an SDS/12%-polyac ylamide gel. A. ch oococcum GS was immunop ecipi a ed wi h 0, 4, 8, 16, 30 and 40 ,u o an i-GS an ibodies (lanes 1-6). Lane 7 co esponds o he an ise um alone (30 ,ul). Molecula masses (M) o p o ein s anda ds a e indica ed. kDa 97.4 66.2 - 45- 1 2 3 4 - - GS Figu e 3 In i o 2P labelling o ammonium-inac i a ed GS om A. ch oococcum 32p labelling o combined ni ogen- ee-g own cells was ca ied ou as desc ibed in he Ma e ials and me hods sec ion. The cell ex ac s hus ob ained (50 jug o p o ein) we e elec opho esed in an SDS/1 0%-polyac ylamide gel and he gel subjec ed o au o adiog aphy. Lanes: 1, cell ex ac o non-ammonium- ea ed cells (con aining ac i e GS); 2, 3 and 4, cell ex ac s om cells ea ed wi h 0.5 mM NH4C0 o 30, 60 and 90 min espec i ely (con aining inac i e GS). The molecula masses (M) o p o ein s anda ds a e indica ed. was subjec o co alen modi ica ion in esponse o ammonium in A. ch oococcum. In i o labelling o A. ch oococcum GS When diazo ophically g own A. ch oococcum cells we e p e- incuba ed in he p esence o H332PO4 and hen subjec ed o an ammonium shock (5 mM NH4Cl), he 59 kDa p o ein p e iously iden i ied as GS subuni was labelled, his labelling inc easing wi h ime o incuba ion in he p esence o ammonium (Figu e 3). This esul indica ed ha GS was modi ied as a consequence o he ammonium shock by a g oup con aining phospha e. I is well es ablished, as s a ed abo e, ha GS om mos G am-nega i e bac e ia is egula ed by an adenylyla ion- deadenylyla ion sys em and ha , in his case, he modi ied ,enzyme is adiolabelled in i o in he p esence o 32P. To check whe he his egula o y p ocess is ope a i e in A. ch oococcum, wo app oaches we e made. Fi s , when cells we e pulse-labelled wi h [3H]adenine, he enzyme was adiolabelled in esponse o _0 Co u) E 0 Co 0 Time (h) Figu e 4 E ec o alkaline phospha ase and phosphodies e ase on glu amine syn he ase Inac i a ed by ammonium Diazo ophically g own A. ch oococcum cells we e ea ed wi h 5 mM NH4CI o 1 h o inac i a e GS and hen used o ob ain he co esponding cell ex ac . The ex ac s we e incuba ed a 37 OC wi h 30 uni s/mg p o ein alkaline phospha ase (O), 1 uni Vmg o p o ein phosphodies e ase (0) o wi hou any addi ion (OJ), and he Mg2+-dependen in i o y-glu amyl ans e ase ac i i y (ac i e o m) was de e mined a he indica ed imes. To ollow he elease o 32p om adiolabelled GS (inse ), he ex ac s om ammonium- ea ed cells we e immunop ecipi a ed a e 1 h incuba ion wi h ei he phosphodies e ase (lane 3), alkaline phospha ase (lane 4) o no addi ion (lane 2). Lane 1 ep esen s he immunop ecipi a e o he ex ac om non-ammonium- ea ed cells; 100% o GS ac i i y co esponds o 300 muni s/mg o p o ein. In he inse M is molecula mass. ammonium addi ion in a simila way o ha desc ibed in Figu e 3 (see below), indica ing ha he modi ying g oup also con ained adenine. Secondly, ea men o an enzyme p epa a ion om ammonium-g own cells, and hence o low ac i i y, wi h snake- enom phosphodies e ase p omo ed he e-ac i a ion o GS ac i i y (Figu e 4). A simila ea men wi h alkaline phospha ase did no media e any e-ac i a ion o GS. Analysis by SDS/PAGE and au o adiog aphy o he enzyme p epa a ion a e e- ac i a ion wi h phosphodies e ase and hen immunop ecipi a ed demons a ed emo al o he modi ying g oup, as shown in Figu e 4 (inse ). Taken oge he hese esul s s ongly suppo ha , in A. ch oococcum, GS is egula ed by an ammonium- induced adenylyla ion-deadenylyla ion. P e en ion by MSX o GS modMca ion MSX is an ex ensi ely used inhibi o o GS ac i i y. In A. ch oococcum cells ha had been p eincuba ed wi h MSX, am- monium was wi hou e ec on ei he ni ogenase ac i i y [2] o ni a e up ake [4], he conclusion being d awn ha ammonium mus be me abolized o exe i s inhibi o y e ec on hese p ocesses. Figu e 5 illus a es an expe imen on [3H]adenine- labelling o GS wi h A. ch oococcum cells subjec o an am- monium shock in he absence (lane 2) and in he p esence o MSX (lane 3). In he con ol ea men (lane 1), he cells we e incuba ed in he p esence o adiolabelled adenine alone. As can be seen, cells ea ed wi h ammonium showed e y high adio- ac i e labelling, as s a ed abo e, and MSX p e en ed GS om being co alen ly modi ied. Though no shown i was ound ha MSX emo ed he [3H]adenine labelling om GS when added once NH4+ inhibi ion o GS had aken place. These esul s he e o e sugges ha o GS adenylyla ion o ake place 644 M. C. Munoz-Cen eno, F. J. Cejudo and A. Paneque M (kDa) 97.4 66.2 45 1 2 3 GS Figu e 5 P e en ion by MSX o in i o [2H]adenine-labelling o GS du ing ammonium Inac i a ion [3H]Adenine labelling o combined ni ogen- ee-g own cells was ca ied ou as desc ibed in he Ma e ials and me hods sec ion. The cell ex ac s hus ob ained (50 ,g o p o ein) we e elec opho esed in an SDS/1 0%-polyac ylamide gel and he luo og am was ca ied ou . Lanes: 1, ex ac om non-ammonium- ea ed cells (con aining ac i e GS); lane 2, cell ex ac om bac e ia ea ed wi h 5 mM NH4CI o 60 min (con aining inac i e GS); lane 3, cell ex ac om bac e ia ea ed wi h 5 mM MSX 30 min p io o he 5 mM NH4CI addi ion. The molecula masses (M) o p o ein s anda ds a e indica ed. M 1 2 3 (kDa)_ 1066- 80- ~-GS 49.5 - 32.5 27.5 18.5 Figu e 6 Wes e n-blo analysis o A. ch oococcum cell ex ac s using an l-GS an ibodies Cell ex ac s we e ob ained om 24 h-old cells g own in combined ni ogen- ee medium (lane 1) and in media con aining ei he 8 mM KNO3 (lane 2) o 10 mM NH4CI (lane 3). Aliquo s (60 9g o p o ein) o cell ex ac s we e elec opho esed in an SDS/12%-polyac ylamide gel, and he Wes e n blo was ca ied ou as desc ibed in he Ma e ials and me hods sec ion. ammonium has o be assimila ed, which is in ag eemen wi h he p oposal ha in En e obac e ia GS adenylyla ion- deadenylyla ion depends ul ima ely on he glu amine/a- oxoglu a a e a io. E ec o he ni ogen sou ce on GS p o ein con en We in es iga ed nex whe he , in A. ch oococcum, he le el o GS p o ein syn hesis was egula ed by ammonium also. To check his possibili y cells ha had been g own unde N2- ixing condi ions, which i is known o exhibi a GS biosyn he ic Mg2+/Mn2+ ac i i y a io highe han in combined ni ogen- con aining medium, indica i e o a low deg ee o adenylyla ion, we e ans e ed o media wi h ni a e o ammonium as he ni ogen sou ce, o again o N2- ixing condi ions. Cells we e hen allowed o mul iply o 24 h, app ox. 10- old he gene a ion ime, and GS p o ein measu ed by Wes e n blo ing. Figu e 6 depic s ha he amoun o GS p o ein in he co esponding cell ex ac s was p ac ically he same independen ly o he a ailable ni ogen sou ce. The e o e, in A. ch oococcum, egula ion o GS ac i i y o e ides p o ein syn hesis con ol. DISCUSSION A he s uc u al le el, GS om mos bac e ia is a dodecame wi h iden ical subuni s. Elec on-mic oscopic s udies and compu e ized image p ocessing ha e shown ha GS is o med om wo supe imposed hexagons, each composed o six subuni s a anged adially wi h espec o he cen al hole [31,32]. I is no su p ising, hen, ha an igenic c oss- eac i i y is de ec ed be- ween A. ch oococcum GS and he an ise um di ec ed agains he enzyme om Synechocys is. I is o be no ed, howe e , ha he an ise um c oss- eac s less e icien ly wi h A. ch oococcum GS han wi h Calo h ix, ano he cyanobac e ium, enzyme [33]. T onick e al. [34] showed ha E. coli GS an ise um showed an igenic homology wi h he GSs o a numbe o G am-nega i e bac e ia, bu no an igenic c oss- eac i i y was de ec ed wi h he GSs o G am-posi i e bac e ia o euka yo ic o ganisms. The only excep ion o his was he c oss- eac ion obse ed be ween E. coli GS an ise um and he G am-posi i e S ep omyces GS. La e on, O e al. [35] held a di e en iew on he basis o he amino acid composi ion, N- e minal sequences and p edic ed con o ma ion po en ials o he GSs om he ilamen ous cyanobac e ium Anabaena sp. s ain 7120, he G am-nega i e bac e ium E. coli, and he g am-posi i e bac e ium Bacillus sub ilis. Since hey obse ed limi ed sequence simila i y among all h ee enzymes, hey p oposed ha an igenic c oss- eac i i y does no necessa ily depend upon he G am eac ion o s a e o adenylyla ion. The esul s p esen ed he e, showing c oss- eac i i y o A. ch oococcum GS wi h an ibody aised agains he cyanobac e ium GS, s ongly suppo his hypo hesis, because he adenylyla ion-deadenylyla ion egula o y sys em is lacking in Synechocys is [19]. Ammonium assimila ion in he azo obac e s is hough o ake place exclusi ely h ough he GS-GOGAT ou e [5,6], and inhibi ion o ei he GS o GOGAT has been widely used in in es iga ions in o he ammonium egula ion o ni ogenase ac i i y o ni a e up ake, as s a ed abo e. On he basis o s udies on he e ec o snake- enom phosphodies e ase ea men on he enzyme isola ed om cells g own on a ni ogen- ich medium, and he in i o inco po a ion o adioac i i y om [14C]ATP in o deadenylyla ed enzyme in he p esence o ei he c ude ex ac om A. inelandii o adenylyl ans e ase om E. coli, i was concluded ha A. inelandii GS is egula ed by adenylyla ion-deadenylyla ion [21]. Expe imen s on in i o in- co po a ion o adioac i i y om ei he [32P]o hophospha e o [3H]adenine, bo h componen s o he modi ying g oup, and he use o an an ibody aised agains GS om a cyanobac e ium, allow us now o show he co alen modi ica ion o Azo obac e GS, ol- lowing an ammonium shock, by he combined me hod o adiolabelling and immunop ecipi a ion. Though ou esul s canno ule ou he possibili y ha GS migh be u he e- ac i a ed by an enzyme ha emo es ADP- ibose, e-ac i a ion o modi ied A. ch oococcum GS wi h snake- enom phospho- dies e ase s ongly sugges ha GS adenylyla ion is o p ime impo ance in he co alen modi ica ion o GS. As men ioned abo e, an ADP- ibosyl ans e ase ac i i y capable o modi ying Rhodospi illum ub um GS in i o has been desc ibed ecen ly [13]. The in e nal C/N balance plays an essen ial ole in he con ol o he ino ganic-ni ogen me abolism o mos bac e ia. In his espec , he cen al ole pe o med by GS is unques ionable. Azo obac e ch oococcum glu amine syn he ase 645 Ac ually, he adenylyla ion o GS is i sel subjec o such C/N balance, as shown by he ac ha inhibi ion o GS by MSX p e en ed he enzyme om adenylyla ion a e ammonium shock. Resul s p esen ed he e sugges ha , in A. ch oococcum, he cen al ole o GS in he con ol o ni ogen me abolism elies only on modula ion o GS ac i i y. Thus Wes e n-blo analysis o c ude ex ac s om cells g own on di e en ni ogen sou ces did no e eal any signi ican in luence o he ni ogen sou ce on he amoun o GS p o ein. In his connec ion i has been epo ed, as men ioned abo e, ha he le el o mRNA o glnA, he gene coding o GS, is no a ec ed by he ni ogen sou ce in A. inelandii ei he [22]. By con as , he GS p o ein le els in ammonium-g own cyanobac e ia a e app ox. 500% o hose ound in cells g own on ni a e [33] o unde N2- ixing condi ions [36]. This wo k was suppo ed by Comisi6n In e minis e ial de Ciencia y Tecnologia, G an B1091/0914 and Plan Andaluz de In es igaci6n G oup 3156. We hank J. C. Reyes his ad ice ega ding expe imen s wi h an i-GS an ibodies. The sec e a ial assis ance o Pepa Pe ez de Le6n is deeply app ecia ed. REFERENCES 1 Laane, C., K one, W., Konings, W., Haake , H. and Veege , C. (1980) Eu . J. Biochem. 103, 39-46 2 Cejudo, F. J., De la To e, A. and Paneque, A. (1984) Biochem. Biophys. Res. 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