Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803

Plant Molecular Biology 27: 789-799, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 789

Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803

Jos~ C. Reyes and Francisco J. Florencio* Departamento de Bioquimica Vegetal y Biologia Molecular and Instituto de Bioqu[mica Vegetal y Fotosintesis, Universidad de Sevilla-CSIC, Apdo. 1113, 41080-Sevilla, Spain (*author for correspondence)

Received 7 July 1994; accepted in revised form 9 January 1995

Key words: cyanobacteria, glnA gene, glutamine synthetase, Synechocystis sp. PCC 6803, light regulation, nitrogen assimilation

Abstract

The glnA gene, encoding type I glutamine synthetase (GS) in Synechocystis sp. PCC 6803, showed a high sequence similarity with other cyanobacterial glnA genes. A dramatic decrease in the amount of glnA mRNA, a single transcript of about 1.6 kb, was observed after transfer to darkness, or after incubation with the electron transport inhibitors D C M U or DBMIB. The levels of glnA transcript were fully recovered after 5 min of reillumination. The glnA m R N A was found to be equally stable both in the light and the dark (half-life about 2.5 min). Unlike the glnA messenger, the amount of GS protein was not reduced in the dark. Synthesis of the glnA transcript in the dark required the presence of glucose. In addition, glnA transcription in a Synechocystis psbE-psbF mutant lacking photosystem II required the presence of glucose even when grown in the light. These observations indicate that glnA transcription is under the control of the redox state of the cell. Finally, nitrogen starvation provoked a delay in the decrease of glnA transcript in darkness, suggesting a connection between nitrogen and redox controls of glnA transcript levels.

Introduction

Glutamine synthetase (GS) catalyzes the ATP- dependent synthesis of glutamine from ammonium and glutamate in an enzymatic reaction that connects carbon and nitrogen metabolism. In photosynthetic eukaryotes and cyanobacteria, ammonium assimilation takes place mainly through the sequential action of glutamine syn-

thetase (GS) and glutamate syntase (GS- G O G A T cycle) [22, 40], requiring two typical photosynthetic products such as ATP and reducing power (ferredoxin or NAD(P)H) [ 13, 40].

In higher plants GS occurs as multiple isozymes, encoded by small gene families, with different patterns of expression in each specific organ [5, 32, 41]. Leaves generally contain one or more cytosolic isozymes (designed GS1), and one plas-

The nucleotide sequence data reported will appear in the EMBL, GenBank and Nucleotide Sequence Databases under the ac- cession number X69199.

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tid isozyme (designed GS2). The plastid-associ- ated form has been shown to be light-regulated. In fact, in etiolated seedlings only GS1 is present but, after transfer to light, synthesis of GS2 is triggered through a transduction signal pathway mediated by the photoreceptor phytochrome [8].

In cyanobacteria, the gene encoding GS type I (glnA) has been cloned and sequenced from several species, including the unicellular Synechococ- cus 7002 [48] and filamentous species such as Anabaena or Calothrix [9, 44]. In all cyanobacteria studied, glnA transcript level is regulated by the nitrogen status of the cell [4, 19, 44, 48], but a light-mediated regulation ofglnA expression has not been reported.

We are interested in the regulation ofglnA gene expression in the facultative heterotrophic unicellular cyanobacterium Synechocystis sp. strain PCC 6803, which has been extensively used as a model in studies on the photosynthetic apparatus [50]. Mutants affected in the photosystem I (PSI) or in the photosystem II (PSII) have been isolated [37, 50], and genes encoding components of the photosynthetic apparatus such as psbA, psbD, psaA or psaB have been shown to be regulated by light, involving or not the photosynthetic electron transport [25, 26, 27, 38]. The D1 polypeptide of PSII is encoded by three different psbA genes in Synechocystis 6803 [16]. The psbA-2 transcript, that represents 90 ~o of the total psbA transcript, is present at low levels in the dark and increases with increasing light intensity, independently of the photosynthetic electron transport [25, 26, 27]. However, the stability of psbA-2 transcript increases in the dark (half-life 8 h versus 7 min) and is controlled by the photosynthetic electron transport [27]. On the other hand, the transcript of the rbcLS operon, encoding the large and the small subunits of the ribulose-bisphosphate carboxylase/oxygenase (Rubisco), is not detected after 60 min in the dark, but mRNA levels remain similar to those observed in the light when glucose is present in the culture medium [27].

In this work we report the sequence of the glnA gene from Synechocystis 6803 and describe the regulation of its expression. We show for the first time in cyanobacteria that glnA gene expression is

regulated, at the transcriptional level, by the redox state of the cell.

Material and methods

Bacterial strains and growth conditions

Synechocystis sp. strain PCC 6803 was grown at 35 °C in B G l l medium [35] under continuous illumination (50W/m) and bubbled with 1.5~o (v/v) CO2 in air. When ammonium was used as nitrogen source, nitrate was replaced by 10 mM NH4C1 and the medium was buffered with 20 mM N-tris(hydroximethyl)methyl-2-aminoethanesul- fonic acid (TES) buffer. Dark conditions were obtained by wrapping the flasks in aluminum foil. For myxotrophic growth, glucose was added to a final concentration of 10 mM. Strain T1297 is a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes are replaced by a kanamycin-resistance gene [30]. This mutant was grown in BG11 medium supplemented with glucose 10 mM and kanamycin 50/~g/ml. Escheri- chia coli DH5~ was used as the host for plasmid preparation. In complementation experiments the glutamine auxotrophic E. coli strain ET6017 [ ara D139 A(argF-lac)205 flbB5 301 pstF25 relA 1 rspL150 A(glnG-A or glnL-A)229 rha-lO deo C1] (E. coli Genetic Stock Center, Yale University) was used. Luria broth was supplemented with ampicillin at 100/~g/ml or kanamycin at 50 #g/ml when required. Complementation experiments were performed in glucose minimal medium [29] supplemented with 40/~g/ml ampicillin.

Sequencing and sequence analysis

Nested deletions were generated with a Nested Deletion Kit from Pharmacia. The complete sequence of both strands of glnA gene was determined by the dideoxy chain termination method [36], using Sequenase 2.0 (USB). Computer analysis was carried out using the sequence soft- ware package of the Genetics Computer Group of the University of Wisconsin [7].

RNA isolation and northern blot analysis

Total RNA was isolated from mid-exponential- phase cultures of Synechocystis 6803 essentially as described by Mohamed and Jansson [26], except that samples of 35 ml were taken and that before lysis cells were frozen in liquid nitrogen and ground in a mortar, while frozen. RNA was denatured, fractionated on 1~o agarose/formal- dehyde gels, and transferred to nylon membranes (Hybond N-plus; Amersham) using a Transvac Vacuum Blotter (Hoefer). 50 mM N a O H was used as transfer solution. Prehybridization and hybridization (5 × SSPE (1 x SSPE=0 .03 M NaC1, 0.01 M NaH2PO4 pH 7.7, 1 mM Na2- EDTA), 5 × Denhardt's solution, 0.5 ~o w/v SDS, 50/~g/ml salmon sperm DNA, 50~o v/v forma- mide) were done at 42 ° C. After hybridization the filters were washed two times for 10 min each in 2 x SSPE, 0.1~o w/v SDS at room temperature and 20min in l x SSPE, 0.1~o w/v SDS at 65 ° C. Relative transcript levels were quantified with a scanning densitometer (Bio Image, Milli- pore Corporation) of at least two different autoradiographs.

The glnA probe was generated by labelling a 631 bp Eco RV-Eco RV internal glnA fragment from pJCR3 [33]. As a control for sample load- ing all the filters were stripped and reprobed with an about 450 bp Hind III-Bam HI fragment from pAV1100 [47] which contains the RNase P RNA gene from Synechocystis 6803.

Determination of glnA-specific mRNA half-fives

In order to determine the effect of darkness on glnA transcript stability, the RNA synthesis of Synechocystis cells either exponentially growing in the light or transferred to darkness was blocked by the addition of rifampicin (400 #g/ml) to the culture; total cellular RNA was isolated at several times there after. The decay of glnA transcript was followed by northern blot hybridization. After densitometric quantification of the signals on the autoradiograms, the half-life of the transcript was estimated from a semi-logarithmic plot of relative transcript level versus time.

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Western blot analysis

One ml samples of Synechocystis cell suspension were taken at the indicated times, in 1 ml micro- centrifuge tubes and the cells were harvested by centrifugation at 4 ° C. After washing in 50 mM HEPES buffer pH 7, the cells were resuspended in 75/~1 of the same buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), and about 40 #1 of glass beads (0.25-0.3 mm di- ameter; Braun) were added. The cells were bro- ken in four consecutive cycles of vortexing for 30 s, each followed by 30 s incubation on ice. Cell debris was removed by centrifugation, and the supernatant was recovered. Samples of cell-free extracts were subjected to S D S-PAGE according to the method of Laemmli [17]. Protein bands were electrotransferred to a nitrocellulose sheet in a BioRad transfer apparatus at 240 mA for 2 h, and western blotting was carried out as previously described [28]. Purified polyclonal mono- specific antibodies obtained against Synechococ- cus sp. PCC 6301 glutamine synthetase were used [21].

Results

Sequence analysis of the Synechocystis 6803 glnA gene

In a previous work we reported the cloning of the glnA gene from Synechocystis 6803 [23]. A 2.5 kb fragment containing the glnA gene (as indicated by its ability to complement an E. coli glnA mutant), was sequenced. A 1431 bp open reading frame that encodes a 476 amino acid protein was found. This polypeptide showed a high amino acid sequence identity with other prokaryotic G S s type I, such as E. coli (54 .7~) or B. subtilis (39.9 ~o ), and specially with those of other cyanobacterial GSs (about 78~o ). An apparent molecular mass of 53468 Da was deduced from the amino acid sequence, in accordance with that of the previously purified Synechocystis 6803 GS [24]. A putative ribosome-binding site, AGGAG, identical to the E. coli consensus, was found 5 bp

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upstream of the coding region. The same sequence is also found 5 bp upstream of the Anabaena and Calothrix glnA ATG start codon [44, 9].

Transcriptional regulation of the glnA gene in response to light-dark transitions

We have previously reported a light-mediated regulation of GS activity in the cyanobacterium Synechococcus sp. PCC 6301 [21]. Inactivation of GS in the dark has also been observed in Syn- echocystis 6803 (manuscript in preparation). To determine if light-dark transitions also affect glnA transcript levels, we isolated total RNA from mid-exponential phase Synechocystis cultures grown either photoantotrophically or myxotrophi- cally, under normal illumination or after 4 h in the dark. In all experiments, nitrate was used as nitrogen source. RNA was then subjected to northern blotting. As shown in Fig. 1, a single glnA transcript of 1.6 kb was detected. The levels of

glnA transcript decreased in the dark in photoautotrophic but not in myxotrophic cultures, suggesting that regulation of glnA transcript level is under redox control. As indicated in Fig. 2A, the glnA transcript levels decreased dramatically after transferring a photoautotrophic Synechocystis culture from light to dark. After 2.5 h of darkness, the culture was returned to standard conditions of illumination; in less than 15 min, the glnA transcript reached levels higher than the steady-state level in the light. Densitometric quantification of the resulting autoradiographs (Fig. 2B) showed that, after 30 min in the dark, the level of glnA transcript decreased 28-fold, and was completely undetectable after 2.5 h of darkness, even after a

Fig. I. Effect of darkness on the expression of the glnA gene from Synechocystis 6083 cells grown with or without glucose. Total RNA was isolated from Synechocystis cells grown in BG11 medium, with or without 10 mM of glucose and before or after 4 h of incubation in the dark. RNA was denatured, electrophoresed in 1 ~ agarose gel, blotted and hybridized with a 631 bp Eco RV-Eco RV glnA probe. 10/~g of total RNA was loaded per lane. The filter was stripped and rehybridized with a RNase P RNA gene probe. Transcript size was estimated by comparison with 23S, 16S and 5S rRNAs [26].

Fig. 2. Time course of glnA transcript levels in the dark, and effect of reillumination. A, Photoautotrophically grown Syn- echocystis cells were transferred to darkness. After 2.5 h, the culture was reilluminated; samples for RNA isolation were taken at the indicated times. RNA was processed and hybridized as in Fig. 1. B. Relative levels of glnA transcript. Values are averages of two hybridization experiments and are expressed as a percentage of the initial value.

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Fig. 3. Effect of darkness on the stability of glnA mRNA. Synechocystis cells were photoautotrophically grown to mid- log phase. At t = 0, cultures were transferred to darkness while others were maintained under standard illumination. At the same time rifampicin (400 #g/ml) was added to the cultures to block transcription. RNA was isolated at several times before and after rifampicin addition. The decay of the glnA transcripts was followed by northern blotting (upper panel). The mRNA levels were quantified by densitometry. Linear regres- sion analysis was performed, and plots were drawn of relative mRNA levels versus time. The half-lives of the transcript were calculated from these plots (bottom panel).

Fig. 4. Effect of darkness and reillumination on the amount of GS protein. A photoautotrophically grown Synechocystis culture was transferred to darkness for 8 h and returned to standard conditions of illumination. At the indicated times, 1 ml samples were taken and subjected to western blot analysis as described in Material and methods.

responsible for the control ofglnA mRNA level in response to the redox state of the cell. However, the amount of GS protein was almost constant under dark conditions or after switching to the light, as was evidenced by western blotting of extracts from light- and dark-incubated cells (Fig. 4).

long film exposure. Five minutes after reillumination, the glnA transcript levels reached 130~o of the initial level. In contrast to the glnA gene, transcript levels of the RNase P RNA gene remained approximately the same under all the conditions tested. In order to investigate whether darkness affects the glnA m R N A stability, we have determined the half-life of the glnA m R N A under light and dark conditions. The decay of transcript level after rifampicin addition was followed by northern hybridizations (Fig. 3), and plots were drawn of relative m R N A level versus time (Fig. 3). The half-life of glnA transcript was about 2.5 min under both light or dark conditions, suggesting that regulation of the glnA gene transcription is

Effect of photosynthetic inhibitors on glnA transcript levels

Two different photosynthetic inhibitors, D C M U (which blocks transfer of electrons between the PSII complex and the plastoquinone pool [42]) and DBMIB (which prevents the oxidation of plastoquinone by the cytochrome bar complex [34]) were used to investigate the electron transport components involved in the regulation of glnA transcription. For this purpose, Synechocys- tis cells grown under photoautotrophic conditions were treated during 1 h with D C M U 5/~M, DBMIB 5 #M or DBMIB 2 0 # M ; total RNA was then isolated. Both inhibitors, at a final con-

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Fig. 5. A. Effects of photosynthetic inhibitors on the glnA transcript levels. Photoautotrophically-grown Synechocystis cells were incubated either in the absence or the presence of 5 #M DCMU, 5 #M DBMIB or 20 #M DBMIB. RNA was isolated after 1 h. RNA was processed and hybridized as in Fig. 1. B. Relative levels of glnA transcript. Values are averages of two hybridizations and are expressed as percentages of the control.

centrat ion of 5/~M, block photosynthet ic electron t ransport , assayed as 02 evolution (data not shown); however , D C M U provoked a higher decrease in glnA t ranscr ipt level than D B M I B (Fig. 5), suggesting that the redox state of the p las toquinone pool could be involved in the regulation of the amount of glnA t ranscr ipt in Syn- echocystis 6803.

glnA transcript levels in a mutant lacking PSII

We have studied the variat ions in the level ofglnA t ranscr ipt in a Synechocystis 6803 mutan t which lacks P S I I (T1297) [30] and therefore is able to grow only in the presence of glucose. Unde r these

Fig. 6. Effect of glucose deprivation on the level ofglnA transcript in psbE psbF deletion-insertion mutant of Synechocystis 6803 (T1297). Synechocystis T1297 cells grown in BGll medium supplemented with 10 mM glucose (control) were transferred to glucose-free medium for 4 h. Glucose 10 mM (final concentration) was then added to the culture. Alternatively 3-O-methyl-D-glucose was added at the same final concentration. Samples were taken at the indicated times for RNA isolation. RNA was processed and hybridized as in Fig. 1.

condit ions, T1297 Synechocystis cells showed the same level o f glnA t ranscr ipt than the wild-type strain. However , 4 h after transferring the cells to glucose-free medium, the glnA t ranscr ipt was al- mos t undetectable (Fig. 6). After glucose addit ion glnA t ranscript levels close to the original were rapidly recovered. Addit ion of 3-O-methyl-D- glucose, a glucose analogue that is t ranspor ted but not metabol ized in Synechocystis [ 10], had no effect on the level of the glnA t ranscr ipt (Fig. 6). These results clearly indicate that the redox power supplied by glucose metabol i sm controls glnA t ranscr ipt accumulat ion.

Interference between nitrogen control and redox control o f g l n A transcript levels

Transcr ip t ional regulation ofglnA by the nitrogen status of the cell has been previously repor ted in other cyanobacter ia [4, 44, 48, 19]. In Synechocys- tis 6803, nitrogen s tarvat ion also induces an in- crease of glnA gene expression (manuscr ip t in

Fig. 7. Effect o f nitrogen deprivation on the decrease of glnA t ranscr ipt levels in the dark. Synechocystis cells grown photo- autotrophical ly with nitrate as ni trogen source were harvested, washed and t ransferred to nitrogen-free m e d i u m or to nitrate- containing med i um for 15 h and then t ransferred to darkness . Samples for R N A isolation were taken after 0, 1 and 10 h o f incubat ion in the dark. 10 and 5 #g o f total R N A were loaded per lane for the NO3- and the - N samples , respectively. R N A was processed and hybridized as in Fig. 1.

preparation). In order to study if the nitrogen status of the cell affects the redox control ofglnA transcription, Synechocystis cells grown with nitrate as nitrogen source and under continuous illumination were harvested, washed and transferred for 15 h to either nitrogen-free or nitrate- containing medium, and then transferred to the dark for 1 or 10h. As shown in Fig. 7, cells starved for nitrogen displayed 30 ~o of glnA transcript 1 h after transfer to dark, in contrast with non-starved cells, which showed undetectable levels. After 10 h of darkness, the glnA transcript was still detected but at very low level in nitrogen- starved cells.

Discussion

In cyanobacteria, nitrogen assimilation is closely linked to the photosynthetic process. Reduced ferredoxin is the electron donor for both nitrate and nitrite reductases and for glutamate synthase (GOGAT), while ATP is needed for ammonium assimilation by GS [13]; thus an appropriate co- ordination between photosynthesis and nitrogen

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metabolism must be achieved. Because the GS- catalyzed reaction is the first enzymatic step con- necting nitrogen and carbon metabolism, it is an obvious candidate to be light-regulated. In this work we report for the first time in cyanobacteria that transcription of glnA gene is under the control of the redox state of the cell.

Cloning of the glnA gene from Synechocystis 6803 was described in a previous work [23]. Amino acid sequence comparisons between the Synechocystis glnA gene and those of other cyanobacteria (Table 1) revealed a high identity (about 78 ~) . However, the identity found between the glnA sequences of the two unicellular species (Synechocystis 6803 and Synechococcus 7002, 78.9~o identity) was not much higher than between the unicellular and the filamentous species (Anabaena 7120 and Calothrix 7601), about 77,8 ~o. We have recently reported the character- iz ation of a second G S gene (glnN) in Synechocys- tis 6803 [33] which, together with the GS genes of Bacteroides fragilis and Butyrivibrio fibrisolvens, identifies a new family of glutamine synthetases. The amino acids identity between glnN and glnA gene products of Synechocystis was less than 20 ~o, similar to that found between glnN and other prokaryotic glnA gene products.

Northern blot analysis showed that the glnA transcript was almost undetectable 30 min after transferring photoautotrophically growing Syn- echocystis cells from light to dark (Fig. 2). A decrease in the glnA transcript level was also observed after treating Synechocystis cells with photosynthetic inhibitors like D C M U or DBMIB (Fig. 5), suggesting that photosynthetic electron

Table 1. Percentage of amino acid identity between glnA genes of different prokaryotic organisms. 1

6803 7002 7120 7601 E. coli B. subtilis

Synechocystis 6803 - 78.9 77.2 77.8 54.7 39.9

Synechococcus 7002 - 74.5 78.3 54.0 41.3 Anabaena 7120 - 89.5 54.7 38.6 Calothrix 7601 - 53.5 38.7 Escherichia coli - 44.8

1 Percentages o f identity were calculated us ing the G A P pro- g ram [7].

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transport controls the level of the glnA transcript. The level o f m R N A of a gene is determined by the frequency of transcription initiation and the rate of decay. Our experiments indicate that control of the amount ofglnA transcript in response to light/ dark transitions is exerted at the transcriptional level, since under both, light and dark conditions, the half-life of glnA mRNA is almost the same (Fig. 3). The fact that levels of glnA transcript were not affected by light-dark transitions in the presence of glucose (Fig. 1) suggests that respiratory electron transport might also control glnA transcription. Unlike the plastoquinone pools of the chloroplast, those of cyanobacteria can be reduced in the dark by oxidation of respiratory substrates such as glucose [2, 15]. This finding, together with other data (for a review, see [12, 31]), indicates that some or all the respiratory electron transport to oxygen relies on the same electron transport components that connect PSII to PSI. Hence, in cyanobacteria the transfer of energy between the two photosystems, which is modulated by the redox state of the electron transport components, can also be influenced by respiratory electron transport. The fact that, in a Synechocystis mutant lacking PSII (strain T1297), glnA transcript levels depend on the presence of glucose but not of a non-metabolizable analogue (3-O-methyl-D-glucose) confirms that the redox state of the cell controls glnA transcription (Fig. 6). In strain T1297, the reducing power is obtained from the glucose catabolism though the oxidative pentose phosphate pathway [39]. NADPH produced by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase is able to reduce ferredoxin by the back reaction of the ferredoxin-NADPH reductase (FNR) or to donate electrons, via a NAD(P)H dehydrogenase, to the respiratory electron transport chain to generate ATP. In the light, ATP can be also generated through the cyclic electron flow around PSI [12]. Therefore, glucose-starved T1297 cells can be expected to be deprived of reducing power but not of ATP, which confirm that the redox state (and not the energy charge) must control glnA transcription.

In natural conditions cyanobacteria are sub-

jected to a regular diurnal light-dark cycle. The amount of GS protein was almost the same after 8 h of darkness indicating that during the night GS is not de novo synthetized but is not degraded. In the dark without glucose, Synechocystis cells are unable to assimilate any nitrogen source, but a very stable GS protein may confer an advantage in order to start to assimilate nitrogen immedi- ately after light on. A transcript accumulation pattern similar to that found for the glnA gene has been reported for the rbcLS genes [26, 27]. This similarity could be expected since Rubisco and GS participate in metabolic processes, assimilation of carbon and ammonium respectively, which require an input of ATP and reducing equivalents produced by the photosynthetic electron transport. The transcriptional factor NtcA of Synecho- coccus 7942 is involved in the regulation of glnA transcription depending on the nitrogen status of the cell [ 19, 45, 46]. NtcA binds to a palindromic sequence upstream of the Synechococcus glnA gene [19]; this sequence is also found upstream of the glnA gene from Synechocystis 6803 (not shown). The BifA protein of Anabaena 7120, which seems to be the NtcA homologue in Ana- baena, binds to the promoter region of Anabaena glnA gene, but also to sequences upstream of the rbcL and xisA genes (XisA is a developmentally regulated, site-specific recombinase required for the rearrangement of nif genes) [6, 49]. Together with the fact that rbcLS and glnA genes display a similar pattern of transcript accumulation, these observations could suggest that NtcA (BifA) not only responds to changes in nitrogen status but also to the redox state of the cell. However we cannot rule out the existence of other DNA- binding factor(s) involved in redox transcriptional regulation.

The fact that nitrogen starvation causes a delay in the decrease of glnA transcription in the dark may indicate an interference between nitrogen and redox controls of glnA transcription. One candidate to play a role in the connection between the regulation of nitrogen metabolism and electron transport is the bacterial regulatory protein Pn (glnB gene product), which has been recently isolated in Synechococcus 7942 [14, 43]. Pn is phos-

phorylated in nitrate-growing cells, in the light, while darkness or the presence of ammonium in the culture medium results in a decrease of its phosphorylation [ 11 ]. In enteric bacteria, PH protein is involved in the regulation of the synthesis and the activity of GS [20]; such an involvement has not been demonstrated in cyanobacteria. On the other hand, nitrogen-starved cyanobacterial cells accumulate carbohydrates [1, 18]; in the dark, these can be mobilized to produce ATP and reducing equivalents that may cause the delay observed in the decrease of the glnA transcript.

In the cyanobacterium Calothrix sp. strain PCC 7601, the redox state of the cell has been proposed as a general metabolic signal involved in differentiation of specialized cells such as het- erocysts and hormogonia [3]. In fact, D C M U partially inhibits heterocyst differentiation, suggesting again an interference between nitrogen and redox controls of gene expression.

In summary, our results demonstrate that transcription of the Synechocystis glnA gene is tightly controlled by the redox state of the cell. Further studies are required to elucidate the regulatory cascade that modulates glnA transcription rate responding to different environmental stimuli in cyanobacteria.

Acknowledgements

We thank Dr H. B. Pakrasi for the gift of the strain T1297, and Dr A. Vioque for providing us the pAV1100 plasmid. The critical reading of the manuscript by Dr J. Casadesus and Dr A. Vioque is also acknowledged. This work was supported by grants from DGICYT (PB91-0127) (Spain) and by Junta de Andalucia.

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Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803