gated, PI, is modified by uridylylation (reviewed by Merrick and. Edwards, 1995). This suggests conservation of the modification mechanism but diversification of ...
Eur. J . Biochem. 244, 869-875 (1997) 0 FEBS 1997
Phosphoprotein PI, from cyanobacteria Analysis of functional conservation with the PI, signal-transduction protein from Escherichia coli Karl FORCHHAMMER and Andrea HEDLER Lehrstuhl fur Mikrobiologie der UniversitAt Miinchen, Miinchen, Germany (Received 19 December 1996/28 January 1997) - EJB 96 1880/1
The signal transduction protein PI, from Escherichia coli is modified by uridylylation, whereas its counterpart from the cyanobacterium Synechococcus PCC 7942 is phosphorylated at a seryl residue. To elucidate functional conservations between these proteins, we compared the Synechococcus PI, protein with the known properties of the E. coli PI, protein. Similar to the E. coli protein, Synechococcus PI, binds the metabolites 2-oxoglutarate and ATP in a mutually dependent manner. The synergism of ligand binding was analyzed in detail. The ATP-binding site of Synechococcus PI, could be labelled with 5’-pfluorosulfonylbenzoyladenosine. By heterologous expression of the cyanobacterial glnB gene in E. coli we showed that Synechococcus PI, can be modified by the E. coli PI, uridylyltransferase. The presence of Synechococcus PI, prevents signal transduction of E. coli PI, to NtrB, presumably by non-functional competition. We therefore propose that the primary function of Synechococcus PI, is to sense 2-oxoglutarate, the carbon skeleton required for nitrogen assimilation.
Keywords: cyanobacteria; Synechococcus; nitrogen regulation ; GlnB ; nitrogen assimilation
The glnB gene product, also termed PI, protein, is a central signal-transduction protein in the nitrogen control of bacteria. A bifunctional uridylyltransferase/uridylyl-removing enzyme uridylylates PI, at a tyrosyl residue under nitrogen-limiting conditions and deuridylylates PI,-UMP during nitrogen abundance. In Escherichia coli, the modification state of PI, regulates the activity of the transcription activator NtrC, via NtrB, and glutamine synthetase activity, via the adenylyltransferase enzyme GlnE (Mangum et al., 1973; Reitzer, 1996). In this organism, the molecular basis for PI, modification and thus for nitrogen sensing has been intensively investigated. Glutamine, 2-oxoglutarate and ATP have long been recognized as regulatory metabolites of the sensing system (Adler et al., 1975). It was shown that glutamine specifically inhibits the uridylyltransferase activity and promotes the uridylyl-removing activity of the enzyme (Atkinson et al., 1994), whereas 2-oxoglutarate and ATP are bound by PI, (Kamberov et al., 1995). It was suggested that PI, could function as sensor of 2-oxoglutarate and ATP ; liganding these metabolites may result in an allosteric alteration of PI, which facilitates the uridylyltransferase reaction. PI,-like proteins have been found in a variety of other organisms. In proteobacteria, Pi, proteins seem to be involved in additional functions to those in E. coli, such as control of N, fixation in Azospirillum brasiliense (de Zamaroczy et al., 1993) or regulation of nitrate reduction in Rhizobium leguminosarum (Amar et a]., 1994). Despite this diversity, in all proteobacteria investigated, PI, is modified by uridylylation (reviewed by Merrick and Edwards, 1995). This suggests conservation of the modification
mechanism but diversification of downstream signalling of PI, in different proteobacteria. In the cyanobacterium Synechococcus PCC 7942, a P,,-like protein has been identified, which was shown not to be modified by uridylylation but rather by phosphorylation at a serine residue (Forchhammer and Tandeau de Marsac, 1994). Ser49 could be identified as the phosphorylation site (Forchhammer and Tandeau de Marsac, 1995b), which is separated by only one amino acid from the conserved tyrosine residue (Tyr51) uridylylated in proteobacteria. PI, phosphorylation in Synechococcus resembles the eukaryotic type of signal transduction involving specific protein-serine-kinase and phosphatase activities. The phosphorylation state of the Synechococcus PI,protein was shown to respond to the nitrogen and carbon supply of the cells. Furthermore, evidence was provided that showed that Synechococcus PI, is involved in balancing nitrogen and carbon assimilation (Forchhammer and Tandeau de Marsac, 1995a). In vitro phosphorylation experiments revealed that phosphorylation of Synechococcus PI,depends on 2-oxoglutarate and ATP; the phosphorylation reaction is not influenced by glutamine. These findings raised the questions of which nitrogen-sensing mechanism is operating in cyanobacteria and what functional conservation in the PI,signalling system is common to cyanobacteria and proteobacteria. In this report, we compared the properties of Synechococcus P,, with those of the E. coli P,, protein. Our investigations reveal functional similarities in the metabolite-sensing mechanism between the cyanobacterial and the proteobacterial PI, signal-transduction systems.
Correspondence to K. Forchhammer, Lehrstuhl fur Mikrobiologie der Universitat Munchen, Maria-Ward-Str. la, D-80638 Miinchen, Germany Fax: +49 89 179198 62. Abbreviation. FSO,BzAdo, 5’-p-fluorosulfonylbenzoyladenosine.
MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains used are listed in Table 1. Cyanobacteria were grown photo-autotrophically in BGI 1 medium (Rippka, 1988) at 30°C
Forchhammer and Hedler (Eur: J. Biochem. 244)
Table 1. Strains of E. coli used in this study. Strain
Relevant genotype or phenotype
F-IendAl recA1 hsdRI7 thi-1 gyrA Hanahan (1983) supE44 relAI A(lacZYA-arg F),, ,hP (m80lucZAMI 5) endA1 ihi-1 hsdR17 supE44 AlncCJ169 Backman et al. (1981) as YMCIO, AglnB Bueno et al. (1985) as YMC10, AglnBAglnL Bueno et al. (1985) as YMC10, glnD: :TnlO Reitzer and Magasanik (1985)
YMCIO RB9060 RB9135 YMC26
as described previously (Forchhammer and Tandeau de Marsac, 1994). For cloning experiments, ligations were transformed in competent E. coli DH5a cells, which were grown in Luria-Bertani medium (Sambrook et al., 1989). For the physiological analysis of E. coli cells expressing the Synechococcus gEnB gene, cells were grown either under nitrogen excess (Luria-Bertani medium containing 0.2% glutamine and 0.4% glucose) or in nitrogen-limiting minimal medium containing either 0.2 % glutamine and 0.4 % glucose or 0.2% arginine and 0.4 % glucose as described by Bueno et al. (1985). Materials. Restriction enzymes and other enzymes required for cloning experiments were obtained from Boehringer Mannheim. Radiochemicals were purchased from NEN-Dupont de Nemours. Chromatographic equipment was from Pharmacia, and fine chemicals were obtained from Sigma or Fluka. Construction of plasmid pMPlB overexpressing the Synechococcus PCC 7942 glnB gene in E. coli. A partial library of 1.5-kb PstI fragments of chromosomal DNA from Synechococcus PCC 7942 was constructed with the pBluescript SK+ vector (Stratagene). Clones carrying the gZnB gene were identified by colony hybridization according to standard procedures (Sambrook et al., 1989) using a 24-residue oligonucleotide (MWG-Biotech) corresponding to a sequence of the Synechococcus PCC 7942 glnB gene (5'-CGGGATGACGGTTTCAGAAGTGCG-3') (Tsinoremas et al., 1991). Plasmids, in which the glnB coding sequence was orientated such that it could be transcribed from the P,n, promotor of the vector were identified by restriction analysis. The resulting plasmid, pMPlB, was checked by sequencing. Overproduction of Synechococcus PI, in E. coli and purification of the protein. E. coli strain RB9060 (AgZnB) was transformed with plasmid pMP1B. Transformants were grown overnight at 37°C in 2 1 Luria broth containing 100 pg/ml ampicillin. Synechococcus PI, was overproduced in these cells, as visualized by SDSPAGE and immunoblot analysis. Cells were harvested by centrifugation (13 g wet cell mass), washed once in 50 mM TrislC1, pH 7.4,50 mM KCI, S mM MgCI, and 1 mM EDTA, and suspended in 40 ml of the same buffer containing 0.5 mM phenylmethylsulfonyl fluoride. Cells were broken by two consecutive passages through a French pressure cell at 110 kPa. During the following purification procedure, the presence of Synechococcus PI, was monitored by immunoblot analysis. Unbroken cells and debris were removed by centrifugation at SOOOXg and 20000Xg, respectively. The cell lysate was cleared by protamine sulfate precipitation [0.2 % (mass/vol.)] and the cleared lysate was fractionated by ammonium sulfate precipitation. The fraction precipitating between 30 % and 50 % ammonium sulfate saturation was collected, suspended in 10 mM potassium phosphate, pH 7.3, and desalted by passage through a Sephadex G25 column equilibrated with the same
buffer. The flow-through was subjected to a heat-precipitation step (6 min at 60"C), and denatured protein was removed by centrifugation. Chromatographic separation on DEAE-Sepharose, phenyl-Sepharose and Superdex 75 were performed as described previously for the purification of PI,from Synechococcus (Forchhammer and Tandeau de Marsac, 1995b). The final yield was 20 mg Synechococcus PI,, which was stored at -20°C. Enzymatic assays and protein determination. Glutamine synthetase activity was determined in permeabilized cells as described by Pahel et al. (1982). The average state of glutamine synthetase adenylylation ( n )was determined as described (Stadtman et al., 1979). Protein determinations in whole cells were carried out by the method of Lowry et al. (1951), protein concentrations in solutions were assayed according to Bradford (1976) by means of the BioRad protein-assay reagent. Quantitation of purified Synechococcus PI, was performed by determining the absorption at 277 nm with a specific absorption coefficient of 2840 M-' cm-', derived from the amino acid sequence of the protein (Tsinoremas et al., 1991). Binding of 2-oxoglutarate and ATP to Synechococcus PI,. Binding of radiolabelled ligands to Synechococcus PI, was measured as described by Kamberov et al. (1995), by means of ultrafiltration through a 10-kDa cut-off membrane (Nanosep 10 k, Pall Filtron) or equilibrium dialysis (custom-made 2 0 0 4 reaction chambers). Binding analyses were carried out in 50 mM Tris/CI, pH 7.4, 100 mM KC1 and 10 mM MgCl,. Synechococcus PI, and ligands were added as indicated. Radiolabelled ligands were quantitated by counting three aliquots for each determination in a Packard Tri-Carb 2100 TR liquid scintillation instrument with Rotoszint eco plus scintillation cocktail (Roth).
5'-p-fluorosulfonylbenzoyladenosine (FS0,BzAdo) labelling of Synechococcus Pll. For labelling of Synechococcus PI, with FSO,BzAdo, 7.4 pg purified protein was incubated in 20 pl 10 mM sodium phosphate, pH 7.4, 50 mM KCI with 0.25 mM or 0.5 mM FS0,-BzAdo (from 10 mM FS0,BzAdo in dimethyl sulfoxide) in the presence or absence of 2.5 mM 2-oxoglutarate, and the reaction was allowed to proceed at 30°C for 20 min. Competition experiments with ATP were carried out under the same conditions, except that 0.25 mM ATP and 5 mM MgC1, were added. The reactions were stopped by the addition of an equal volume of 2Xconcentrated SDS sample buffer and loaded on a 15 % SDS/polyacrylamide gel (Laemmli, 1970). After electrophoresis, proteins were blotted on nitrocellulose (Schleicher & Schiill, BA-S 85) and FS0,BzAdo-labelled Synechococcus PI, protein was visualized by means of FS0,BzAdo-specific antibodies and a chemoluminescent detection system (Boehringer Mannheim). Analysis of Synechococcus PI, modification. For the analysis of PI, modification, the different mob es of modified and unmodified protein in non-denaturing polyacrylamide gels was investigated following the procedure described previously for the analysis of P,, phosphorylation in Synechococcus (Forchhammer and Tandeau de Marsac, 1994), which also works for the analysis of PI,uridylylation in E. coZi (Atkinson et al., 1994). In vivo ['H]uracil labelling of Synechococcus PII. Cells were grown in Luria-Bertani medium to an A,,, of 0.4, then the cells were transferred to nitrogen-deficient minimal medium containing 0.2% arginine, 0.4% glucose, and a 1 ml aliquot of cell suspension (A,,, = 0.4) was incubated at 37°C for 90 min in the presence of I 0 pCi [?H]uracil (40 Ci/mmol). Cells were harvested by centrifugation, resuspended in 200 p1 50 mM Tris/ CI, pH 7.4, 5 mM EDTA and broken by sonification. From the crude extract, Synechococcus PI, was recovered by immunoprecipitation as described previously (Forchhammer and Tandeau de Marsac, 1995b). The immunoprecipitates were analyzed by liquid scintillation counting as described above.
Forchhammer and Hedler (Eur: J . Biochem. 244) 3.0
[Bound 2-oxoglutarate] (vM)
Fig. 1. Binding of 2-oxoglutarate to Synechococcus P,,. Binding analysis was performed by the ultrafiltration technique. The concentration of Synechococcus PI, trimers in the experiments was 1.7 mM. (A) Binding of 2-oxoglutarate to Synechococcus PI, in the presence of 2.5 mM ATP. 2['4C]oxoglutarate was added (1-40 pM). The data are shown as a Scatchard plot; B/F designates the ratio of protein-bound ligand (B)/free ligand (F). The regression line through the data points was calculated by means of the Sigma-Plot program. From the slope of the plot, the apparent dissociation constant Kd was calculated as 3.8 pM. The stoichiometry of binding ( n ) was 1.42 mol 2-oxoglutarate/Pll trimer. (B) Dependence of 2oxoglutarate binding to Synechococcus P,, on ATP. Binding of 2-oxoglutarate to Synechococcus PI, was analyzed by Scatchard-plot analysis as described in (A), in the presence of different concentrations of ATP (0.025 mM: Kd = 6.09 pM, n = 0.72; 0.05 mM: Kd = 5.32 pM, n = 0.95; 0.1 mM: Kd = 4.48 pM, n = 1.1; 0.5 mM: K,, = 3.98 pM, n = 1.31; 1.25 mM: Kd = 3.87 pM, n = 1.35; 2.5 mM: K,, = 3.80 pM, n = 1.42). The apparent association constants (K., = UKd) are plotted against the corresponding ATP concentration. (C) Double-reciprocal plot of the data in (B). The regression line was fitted by means of the Sigma-Plot program. From the plot, an apparent K, for ATP to stimulate 2-oxoglutarate binding to Synechococcus PI, can be calculated according to Lineweaver and Burk (1934) ( K , = 17 pM, K,(max) for 2-oxoglutarate binding = 3.74 yM).
RESULTS Expression of the Synechococcus PCC 7942 glnB gene in E. coli RB9060 and purification of Synechococcus P,'. PI, had previously been purified from wild-type cells of Synechococcus PCC 7942. However, the yields were not sufficient to perform ligand-binding analysis. To obtain sufficient amounts of Synechococcus PI,, the glnB gene from Synechococcus PCC 7942 was overexpressed from plasmid pMPlB (see Materials and Methods) in E. coli RB9060, which lacks its own PI, protein. Plasmid pMP1B directs the overproduction of a 13-kDa protein, which could be identified as the Synechococcus PI, protein by immunoblot analysis (Fig. 3 A). The overproduced Synechococcus P,, protein was purified to electrophoretic homogeneity following the procedure developed for the purification of Synechococcus P,, from Synechococcus PCC 7942 (Forchhammer and Tandeau de Marsac, 1995b). To analyze whether recombinant Synechococcus PI,exhibits the same biochemical properties as the naturally occuring protein, an in vitro phosphorylation assay with extracts from the PI,-deficient Synechococcus PCC 7942 strain MP2 was performed. Protein phosphorylation was monitored by visualizing the increase in electrophoretic mobility of phosphorylated Synechococcus P,, according to the previously described procedure (Forchhammer and Tandeau de Marsac, 1994). Synechococcus PI, produced in E. coli can be phosphorylated in vitro in a 2-oxoglutarate-dependent reaction and migrates on non-denaturing gel elctrophoresis in the same way as Pi, purified from Synechococcus PCC 7942 (data not shown).
Metabolite binding studies with Synechococcus P,,. The binding of metabolites to Synechococcus PI, was investigated by equilibrium dialysis or by ultrafiltration with 14C-labelled2-0x0glutarate or 'H-labelled ATP as ligands. By these methods, ligand binding with dissociation constants approximately equal to or lower than the concentration of the target protein can be measured. As has been shown for E. coli PI,,binding of 2-oxoglutarate to Synechococcus Pi, could be detected but was strictly dependent on the presence of ATP (Fig. 1). Dissociation constants were calculated by Scatchard-plot analysis of the binding data. In the presence of 2.5 mM ATP, a dissociation constant for 2oxoglutarate binding of 3.8 pM was determined (Fig. 1 A). No 2-oxoglutarate binding could be measured in the absence of ATP. To test the effect of different ATP concentrations on the highaffinity binding of 2-oxoglutarate to Synechococcus PI,,dissociation constants for 2-oxoglutarate were determined in the presence of various concentrations of ATP. Fig. 1 B shows the dependence of the affinity of Synechococcus P,, for 2-oxoglutarate binding as a function of the ATP concentration in the assay. A strong decrease of 2-oxoglutarate binding (association constant K, = UK,,) was observed below ATP concentrations of 0.5 mM. The dependence of 2-oxoglutarate binding to Synechococcus PI, on the presence of ATP shows a Michaelis-Menten-like saturation curve. Transformation of the data to a double reciprocal plot of l/Ka = Kd versus l/[ATP] (Fig. 1C) results in an apparent K,,, of the ATP effect of 17 pM and a K,(max) for 2-oxoglutarate of 3.74 pM.
Forchhammer and Hedler (Eur: .I. Biochem. 244)
[ATP bound] (pM)
[ATP bound] (pM)
1 0 1 2
Fig.2. Binding of ATP to Synechococcus P,,. Analysis of binding of [3H]ATP to Synechococcus PI, was performed in an equilibrium-dialysis apparatus. (A) Binding of ATP to Synechococcus P,, in the absence of 2-oxoglutarate. The binding reactions were performed in the presence of 11.25 pM Syneclzococcus PI,; ATP was added to 2-320 pM. The binding data are shown as a Scatchard plot as described in the legend to Fig. 1. Apparent KC1 = 37 pM, n = O.88/PIl trimer. (B) Scatchard plot of the binding of ATP to Synechococcus P,, in the presence of 2 mM 2-oxoglutarate. 0.2-10 pM labelled ATP was added to 0.75 pM Synechococcus Pll. Apparent K, = 0.5 pM, n = 1.57/P1,trimer. ( C ) Dependence of ATP binding to Synechococcus PI, on 2-oxoglutarate. Binding of ATP to Svnechococcus PI, was analyzed in the presence of 2-oxoglutarate (0 mM: K, = 37 pM, n = 0.88; 0.1 mM: K, = 8.6 pM, n = 1.4; 0.5 mM: Kd = 2 pM, n = 1.5; 1 mM: K, = 1 pM, n = 1.5; 2 mM: K, = 0.5 pM, n = 1.6; 2.5 mM: K, = 0.53 pM, n = 1.7; 5 mM: K , = 0.43 pM, n = 1.9; 10 mM: K, = 0.38 pM, n = 1.9). The apparent association constants were plotted against the corresponding 2-oxoglutarate concentrations. (D) Double-reciprocal plot of the data in (C), as described in Fig. 1. Apparent K,. = 3.9 mM, K,(max) for ATP binding =0.22 pM.
With ATP as a labelled ligand, weak binding to Synechococcus PI, became evident in the absence of 2-oxoglutarate at the limit of detection, with a calculated Kd of 37 pM (Fig. 2A). In the presence of 2 m M 2-oxoglutarate, binding of ATP was strongly stimulated and an apparent Kd of 0.5 pM was calculated (Fig. 2B). Similar to the strategy described above, we analyzed the dependence of the high-affinity ATP binding on the presence of 2-oxoglutarate. Therefore, Scatchard-plot analysis of ATP binding in the presence of 0.1, 0.5, I , 2, 2.5, 5 and 10 mM 2oxoglutarate were carried out. The resulting association constants of ATP as a function of the 2-oxoglutarate concentration are shown in Fig. 2C. A Michaelis-Menten-like saturation curve was observed. However, the 2-oxoglutarate concentration required to saturate ATP binding was considerably higher. A double-reciprocal plot resulted in an apparent K,, for 2-oxoglutarate of 3.9 mM and a Kd(max) for ATP-binding of 0.22 pM (Fig. 2D). To analyze the specificity of nucleotide binding to P,,, binding of 0.5 pM labelled ATP to 1.1 pM Synechococcus PI, (trimers) in the presence of 2 mM 2-oxoglutarate was competed with 50 pM of unlabelled ATP, ADP, GTP, CTP or UTP. As expected, competition with unlabelled ATP reduced ['HIATP binding by 97%, whereas the presence of a 100-fold excess of unlabelled ADP reduced ATP binding by only 31 %. GTP, CTP or UTP could not compete with ATP binding (data not shown).
analog FS0,BzAdo. FS0,BzAdo is an alkylating agent which reacts with amino acids in the vicinity of ATP-binding pockets (Colman et al., 1977). Proteins labelled with FS0,BzAdo can be detected by imniunoblot analysis with anti-FS0,BzAdo-specific Ig. Synechococcus PI, was incubated with 0.5 mM or 0.25 mM FS0,BzAdo in the absence or presence of 2.5 mM 2-oxoglutarate. Synechococcus PI, was labelled by FS0,BzAdo and the degree of labelling was stimulated in the presence of 2-oxoglutarate 1.9-foid, as determined by densitometric quantitation of the immunoblot. To test whether FS0,BzAdo specifically binds to the ATP-binding site of Synechococcus PI,, the same labelling experiments were performed in the presence of 0.25 inM MgATP as competing agent. In the presence of 2-oxoglutarate, ATP reduced FS0,BzAdo labelling by 75%, whereas in its absence the reduction was only 20 %. This indicates that 2-oxoglutarate increases the specificity of Synechococcus PI, towards ATP.
Modification of Synechococcus P,, by uridylylation in E. coli. Pl,-like proteins in proteobacteria are uridylylated at a conserved tyrosine residue. This residue is conserved in Synechococcus P,, (TyrSl), although Synechococcus PI, is not uridylylated in Synechococcus PCC 7942 but phosphorylated at a seryl residue nearby (Ser49). To test whether Synechococcus P,, can function as a substrate of the uridylyltransferase/uridylyl-removing enzyme (GlnD) in E. coli, the Synechococcus PCC 7942 glnB gene 5'-p-fluorosulfonylbenzoyladenosinebinding to Synechococ- was expressed in the E. coli glnD-wild-type strain YMCIO and cus PI1.The nucleotide-binding properties of Synechococcus PI, in the glnD-mutant strain YMC26. Cells were grown in Luriawere investigated by labelling the protein with the Mg . ATP Bertani medium containing excess nitrogen, in which GlnD
Forchhammer and Hedler (Eu,: J. Biochern. 244)
1 2 3
4 5 6
B I 1
Table 2. Effect of Synechococcus P,, on the regulation fo glutamine synthetase (GS) in E. coli strains deficient in components of the PI, signal-transduction cascade. Values for adenylation of GS represent the average number of adenyl groups/GS dodecamer. n.d., not determined. Strain
Fig. 3. GlnD-dependent modification of Synechococcus P,, in E. coli. Immunoblot analysis of the mobility of Synechococcus P,, in non-denaturing PAGE. (A) Cells of E. coli strain YMCIO (glnD') transformed with plasmid pMPlB (lanes 1 and 4) or with pBluescript SK(+) (lanes 3 and 5 ) and cells of E. coli strain YMC26 (glnD-) pMP1B (lanes 2 and 6) were grown in nitrogen-excess Luria-Bertani medium. Aliquots were shifted to nitrogen-limiting minimal medium (Garg; lanes 4-6) for 2 h. Cell-free extracts were prepared in 50 mM Tris/CI, pH 7.4 and 5 mM EDTA. Proteins were separated by non-denaturing PAGE, and the migration of Synechococcus PI, was analyzed by immunoblot analysis as described previously (Forchhammer and Tandeau de Marsac, 1994). (B) Enzymatic treatment of modified Synechococcus P,,. Cell-free extracts of E. coli strains YMCIO pMP1B (I) and YMC26 pMP1B (11) grown under nitrogen-deficient conditions [corresponding to lanes 4 and 6 in (A)] and of Synechococcus PCC 7942 cells grown in the presence of nitrate and thus containing the phosphorylated forms of Synechococcus PI, (111) were treated with phosphodiesterase (lanes 3) or alkaline phosphatase (lanes 4).As a control, the extracts were incubated under the same conditions in the absence of enzyme (lanes 2) or without incubation (lanes 1). The reactions were carried out as described previously (Forchhammer and Tandeau de Marsac, 1994).
should display uridylyl-removing activity. After the removal of aliquots, the cells were transferred to nitrogen-deficient medium and incubated for 90 min to trigger the uridylyltransferase activity. Extracts were prepared, and the electrophoretic mobility of Synechococcus PI, in non-denaturing gels, which depends on its modification, was analyzed by immunoblot analysis. No modification of Synechococcus P,, was observed in nitrogen-excess medium (Fig. 3 A), but under nitrogen-deficient conditions faster migrating forms of PI, appeared in the glnD-wild-type strain, not in the glnD-mutant strain. The additional band appearing above Synechococcus PI, is probably caused by an unspecific reactivity of the antiserum with an E. coli protein, since this band showed up in control lanes lacking Synechococcus PI,. To prove whether the faster migrating forms that appeared in the glnD-wild-type strain could be caused by midylylation of Synechococcus PI,, the extracts were incubated with phosphodiesterase or alkaline phosphatase, since uridylylated forms should be sensitive to phosphodiesterase and serine-phosphorylated forms to alkaline phosphatase treatment (Fig. 3 B). The modification of Synechococcus PI, produced by the glnD-wild-type strain could be removed by phosphodiesterase treatment, suggesting that the protein is uridylylated. As a control, an extract of YMC26 (glnD-) containing unmodified Synechococcus PI, and of Synechococcus PCC 7942 containing the phosphorylated forms of Synechococcus PI,were incubated under the same conditions. The phosphorylated forms of Synechococcus PI, were removed by alkaline phosphatase treatment. To prove that Synechococcus PI, is modified by uridylylation, cells of strains YMClO (wild-type), YMC26 (glnD-) and RB9060 (glnB-) carrying either plasmid pMPlB or pBluescript I1 SK(+) were radiolabelled with [?HIuracil for 90 min after transfer to nitrogen-deficient medium. Extracts of the labelled cells were prepared and Synechococcus PI, was purified by immunoprecipitation and analyzed by liquid scintillation counting. The immunoprecipitates prepared from extracts of the vector controls contained only background radio-
Specific activity of GS
Adenylation of GS
nmol moll y-glutamyl- dodecamer hydroxamte formed .min-' .mg-' YMCIO pBluescript
nitrogen limiting excess nitrogen
nitrogen limiting excess nitrogen
nitrogen limiting excess nitrogen
RE9060 pMB 1B
nitrogen limiting excess nitrogen
nitrogen limiting excess nitrogen
nitrogen limiting excess nitrogen
nitrogen limiting excess nitrogen
n. d. n. d.
nitrogen limiting excess nitrogen
n.d. n. d.