In Vivo Regulation of Glutamine Synthetase Activity in the Marine Chlorophyll b-Containing Cyanobacterium Prochlorococcus sp. Strain PCC 9511 (Oxyphotobacteria)

Abstract

The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light- and nutrient-limited environment.

Prochlorococcus is a marine photosynthetic prokaryote ubiquitous in most intertropical areas of the oceans and is responsible for a significant part of the global primary production (for a review, see reference 27). Its unusual photosynthetic apparatus (12, 13, 15, 29) and wide genetic diversity have attracted increasing scientific interest in recent years. Its ability to tolerate a very wide light gradient has been linked to the co-occurrence in the field of two ecotypes with distinct irradiance optima for growth and photosynthesis (21, 40). While many reports have described these features in detail, little is known about other important aspects of Prochlorococcus metabolism, such as nutrient assimilation. In particular, the nitrogen assimilatory pathways in Prochlorococcus have not yet been studied. However, it is widely accepted that nitrogen is the main limiting nutrient in the upper layer of the oceans (27), and an understanding of the mechanisms involved in nitrogen assimilation could thus provide some keys to unveiling the remarkable ability of Prochlorococcus to colonize very oligotrophic regions. Nevertheless, such studies with Prochlorococcus face two problems: first, this organism is not easy to cultivate, and second, most isolated strains or clones contain contaminant heterotrophic bacteria. Only very recently, Rippka and coworkers described the first axenic strain, PCC 9511 (31), a typical high-light-adapted Prochlorococcus ecotype, allowing proper study of nonphotosynthetic metabolic pathways.

In the present work, we have studied the physiological response of glutamine synthetase (GS) in cultures of Prochlorococcus subjected to different conditions by measuring transferase and biosynthetic activities and antigen concentration. The standard nitrogen assimilatory pathway in non-nitrogen-fixing cyanobacteria is composed of a complex, highly modulated system of transporters, enzymes, and regulatory proteins (6). It allows the use of nitrate, nitrite, ammonia, or urea as a nitrogen source, among others. Urea and ammonia have routinely been used as nitrogen sources for Prochlorococcus (27, 31). GS catalyzes ammonium incorporation into glutamate and is responsible for most ammonium assimilation in photosynthetic organisms, including cyanobacteria (6). The GS-glutamate synthase cycle plays a crucial role in nitrogen assimilation, since most forms of nitrogen are first converted into ammonium before being further metabolized (6).

Here, we report on GS regulation in Prochlorococcus in order to understand its physiological behavior and how it is affected by nutrient limitation in the field. For this purpose, we subjected cultures of PCC 9511 to different conditions, focusing our attention on two parameters known to affect GS regulation: the nitrogen source and the energy state of cultures. We studied the effects of nitrogen and light limitations, as well as the effects of different nitrogen sources and of specific inhibitors blocking several metabolic steps of photosynthesis and nitrogen assimilation.

(Some of the results reported here were obtained during the Second International Workshop on Prochlorococcus, held in Roscoff, France, in 1999.)

MATERIALS AND METHODS

Strains and culturing.

Prochlorococcus sp. strains PCC 9511 (high irradiance adapted, axenic) and SS120 (low irradiance adapted) were routinely cultured in Nalgene polycarbonate flasks (10 liters) using PCR-S11 medium as described by Rippka and coworkers (31). The seawater used as a basis for this medium was kindly provided by the Station Biologique de Roscoff (Roscoff, France) and the Centro Oceanográfico de Fuengirola (Málaga, Spain). Cells were grown in a culture room set at 24°C under continuous blue irradiance (40 and 4 μE m² s⁻¹ for PCC 9511 and SS120, respectively). All experiments were performed during the exponential phase of growth. Growth was determined by measuring the absorbance at 674 nm (A₆₇₄) of cultures.

In vivo experiments.

Aliquots (250 ml) of cultures were taken at various times and centrifuged at 30,100 × g for 5 min in an Avanti J-25 Beckman centrifuge equipped with a JA-14 rotor. After most of the supernatant was poured off and the remaining medium was carefully pipetted out, the pellet was directly resuspended in 500 μl of cold 50 mM Tris-HCl (pH 7.5) and immediately frozen at −20°C until used for enzymatic or immunochemical analysis.

For experiments with different nitrogen sources, standard cultures (growing on ammonium) were harvested as described above, washed twice in nitrogen-free PCR-S11 medium, and resuspended in the same nitrogen-free medium. Aliquots were transferred to new bottles, and the corresponding nitrogen sources [KNO₃, KNO₂, (NH₄)₂SO₄, urea, glutamine, and alanine] were added at a final nitrogen concentration of 400 μM. For experiments requiring darkness, culture bottles were completely covered with two layers of aluminum foil, and the sampling was performed in the dark. For experiments with inhibitors, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) was dissolved in methanol prior to addition to the cultures. Cells from samples to which inhibitors had been added were washed with PCR-S11 medium prior to freezing to avoid the direct effect of the inhibitors (methionine sulfoximine [MSX], in particular) on the GS transferase assay.

Enzymatic assays.

GS transferase activity was determined as previously described (11) over 30 min at 37°C. The composition of the reaction mixture was changed slightly after characterization of the GS transferase activity in Prochlorococcus (S. El Alaoui, J. Diez, and J. M. García-Fernández, unpublished data) and contained the following: 100 mM glutamine, 10 mM sodium hydroxylamine, 50 μM manganese chloride, 10 μM ADP, and 50 mM sodium arsenate in 0.2 M morpholinepropanesulfonic acid (MOPS; pH 7). GS biosynthetic activity was determined basically as described by Marqués et al. (18). One unit of activity is the amount of enzyme that transforms 1 μmol of substrate/min.

After samples were thawed, they were centrifuged at 16,100 × g for 5 min; the supernatants were used for the GS assay. The chlorophyll concentration was determined (17) and used to standardize the enzymatic activities. Error bars shown in all figures correspond to standard deviations for two or three independent experiments.

Immunochemical techniques.

Electroimmunoassay (EIA, or rocket immunoelectrophoresis) was performed essentially as described by Axelsen and Bock (1). Anti-GS rabbit antibodies (300 μl) from Synechocystis sp. strain PCC 6803 (produced as described previously [16]) were added to 15 ml of a solution containing 40 mM Tris (pH 8.6), 100 mM glycine, 600 mM calcium lactate, 1.5 mM sodium azide, and 1% agarose, previously melted and kept at 56°C for 15 min; this mixture was used to prepare the electrophoretic bed on a GelBond (FMC Bioproducts) film sheet. Due to the low GS concentration in crude extracts prepared for enzymatic activity determinations (see above), the extracts for EIA were concentrated fivefold (by resuspending the cells from 250 ml of culture in 100 μl of the same buffer instead of 500 μl). Ten microliters of extract prepared in this manner was loaded into each well. Electrophoresis was carried out overnight with a flat-bed apparatus (FBE 3000; Pharmacia) at 200 V and 10°C. Plates were washed with 15 mM NaCl for 48 h to remove non-cross-reacting proteins and then stained with Coomassie blue to detect immunoprecipitates. The chlorophyll concentration was used to standardize the measured values of the rocket areas.

RESULTS

Adaptation and setting of Prochlorococcus cultures.

The Prochlorococcus strains used in this work were provided by the Station Biologique de Roscoff (Roscoff, France) and the Institut Pasteur (Paris, France). The considerable difficulty in obtaining laboratory cultures of Prochlorococcus resulted in the need for a long period of adaptation of the strains to the conditions of our culture room (several months) prior to experimental work. After an initial period of growth in the original medium (using seawater from Roscoff), the cells were progressively adapted to PCR-S11 medium prepared with oligotrophic seawater from the Mediterranean Sea (near Málaga, Spain), where the final cultures were established. The growth rate was measured by determining the A₆₇₄ (i.e., the red maximum in absorption spectra for Prochlorococcus), which was recently shown to be well correlated with the Prochlorococcus cell concentration (3); in fact, this correlation proved better than that of A₇₅₀ and cell concentration (data not shown). Under our culture conditions, a maximum A₆₇₄ of 0.2 was achieved in carboys of up to 20 liters, with a yield of ca. 100 mg of cells (fresh weight) per liter of culture; thus, physiological experiments could be performed with sufficient protein concentrations for enzymatic assays. Cultures with an A₆₇₄ of ca. 0.05 (exponential phase) were used to start all the experiments.

Nitrogen-mediated regulation.

Exponentially growing Prochlorococcus strain PCC 9511 cultures in standard PCR-S11 medium (i.e., with ammonium as the sole nitrogen source) showed enzymatic activities of 2.5 to 5 U · mg of chlorophyll⁻¹ for GS. Most of the total GS activity (>95%) in crude thawed extracts was detected in the supernatant after 5 min of centrifugation at 16,100 × g, indicating that GS is a soluble enzyme (data not shown). The biosynthetic activity was determined as well. A value of ca. 0.034 U · mg of chlorophyll⁻¹ was obtained; this value is ca. 78-fold lower than the transferase activity.

The effect on the GS level of transferring cultures from ammonium to different nitrogen sources was investigated. Given the loss of cells during centrifugation (due to the extremely small size of Prochlorococcus), the control sample (ammonium) was also subjected to centrifugation and resuspended in standard PCR-S11 medium. Table 1 shows that GS transferase activity was decreased in Prochlorococcus strain PCC 9511 when cells growing on ammonium were transferred to nitrogen-free or nitrate-, nitrite-, or Gln-containing PCR-S11 medium, while it was increased when cells were transferred to Ala- or urea-containing medium. Similar results were observed for the determination of GS biosynthetic activity (Table 1) in these samples, indicating the same trends as in transferase activity. Hence, as far as GS activity is concerned, transfer to nitrate or nitrite has an effect similar to that of nitrogen starvation. Moreover, no growth of PCC 9511 was seen with nitrate or nitrite as the sole nitrogen source (data not shown), even when the molybdenum concentration was increased to 5 mM to ensure that molybdenum cofactor synthesis was not limited. These results confirm a previous report suggesting that the studied Prochlorococcus strain can use only reduced forms of nitrogen, such as ammonium or urea (31).

TABLE 1.

Effect of the nitrogen source on GS activity^a

N source (μM)	GS activity (U/mg of chlorophyll)
	Transferase	Biosynthetic
NH4+	2.70	0.0345
KNO3 (400)	1.74	0.0206
KNO2 (400)	1.60	0.0120
None	2.32	ND
Gln (200)	2.12	0.0199
Ala (400)	4.70	0.0928
Urea (200)	4.76	0.0591

^aInitial cultures of Prochlorococcus PCC 9511 grown on ammonium were centrifuged. The cells were washed with nitrogen-free medium and transferred to new medium containing the indicated nitrogen source. After 24 h, samples were collected and used to determine GS transferase and biosynthetic activities as described in Materials and Methods. ND, not determined. 

The unexpected reduction of GS activity under conditions of nitrogen starvation in Prochlorococcus strain PCC 9511 led us to study this issue further. Figure 1 shows the time course of GS activity in PCC 9511 cultures transferred to nitrogen-free medium. GS activity remained unaffected during the first few hours and showed a slight decrease after 1 day.

FIG. 1.

Effect of nitrogen starvation on GS activity in Prochlorococcus strain PCC 9511. Samples were collected from a 10-liter culture growing on 400 μM ammonium at time −1, and the cells were resuspended in nitrogen-free PCR-S11 medium at time zero. Samples were taken at the indicated times for GS activity determinations. GS activity at time zero was 2.7 U · mg of chlorophyll⁻¹.

Long-term experiments were performed in order to further analyze the response of GS under nutrient limitation. Since phosphorus has been proposed to be the limiting nutrient in some oligotrophic areas of the oceans where Prochlorococcus thrives, such as the eastern Mediterranean Sea (14, 27), we compared the effects of nitrogen versus phosphorus starvation. Figure 2 shows the time course of GS activity in samples of Prochlorococcus strain PCC 9511 cultures subjected either to N or to P starvation and in the corresponding controls grown in standard PCR-S11 medium. GS activity in nitrogen-starved samples showed only minor changes compared to the controls; on the contrary, phosphorus limitation induced a marked decline in GS activity, with only 8% of the initial activity being seen after 5 days.

FIG. 2.

Long-term effect of N versus P starvation in Prochlorococcus. A Prochlorococcus strain PCC 9511 culture was centrifuged. The cells were washed with N- or P-free medium, divided into four fractions, and then resuspended in the following media: standard PCR-S11 (control for N starvation) (▪), N-free PCR-S11 (□), standard PCR-S11 (control for P starvation) (●), and P-free PCR-S11 (○). GS activity at time zero was 1.22 U · mg of total protein⁻¹.

The effect of blocking nitrogen assimilation on GS regulation was studied using two specific enzyme inhibitors (MSX, an inhibitor of GS, and azaserine, an inhibitor of glutamate synthase); these enzymes act consecutively in the nitrogen assimilation pathway.

Figure 3 shows the evolution of the activity after 1 mM MSX was added to PCC 9511 cultures. We found partial inhibition after 24 h; the enzyme was rapidly inactivated (70% inhibition) after only 1 h. A lower inhibitor concentration (100 μM) also was tested; it provoked less than 50% inhibition (data not shown). The addition of azaserine had a different effect (Fig. 3): there was a quick increase in GS activity after 1 h (more than 100%), which stabilized after 8 h until the end of the experiment.

FIG. 3.

Time course of GS inactivation after the addition of MSX and azaserine to Prochlorococcus strain PCC 9511 cultures. Symbols: ▪, control (no addition); ●, addition of 1 mM MSX; ▴, addition of 100 μM azaserine. Inhibitors were added at time zero. GS activity at time zero was 3.27 U · mg of chlorophyll⁻¹.

Regulation through the energy state of cells.

The GSs of most photosynthetic organisms are regulated by light (6). Hence, the effect of subjecting cultures to darkness or to the addition of specific inhibitors of electron transport, such as diuron [3-(3-4-dichlorophenyl)-1,1-dimethylurea; DCMU] and DBMIB, was investigated.

Figure 4 shows the effect of darkness on GS activity in Prochlorococcus strain PCC 9511 cells. The GS level remained almost unchanged even after 24 h with no light. This is a very unusual response, as darkness promotes the downregulation of GS in most other studied cyanobacteria (6, 19, 32). Since it has been shown for Synechococcus sp. strain PCC 6301 that darkness-promoted inactivation is a labile process (19) that reverse when cells are disrupted prior to an enzymatic assay, we checked this possibility for Prochlorococcus by assaying the cells directly after harvesting (without freezing) (data not shown). However, we detected no clear inactivation even under such conditions. Furthermore, we have observed that another strain, Prochlorococcus strain SS120, can be kept alive for more than 1 year under darkness and still show its characteristic green color (data not shown).

FIG. 4.

Effect of darkness on GS activity. A Prochlorococcus strain PCC 9511 culture growing in light was divided into two fractions; one was kept under the same irradiance (control, ▪), and the other was placed in darkness (●). GS activity at time zero was 3.3 U · mg of chlorophyll⁻¹.

Although the absence of light per se had no effect on GS activity, we explored whether the electron flow was involved in GS regulation. This possibility was assessed using DCMU and DBMIB, two inhibitors that block the electron transport chain before and after the plastoquinone pool, respectively (30, 39). Figure 5 shows the time course of GS activity in Prochlorococcus strain PCC 9511 after DCMU was added. A marked decrease (more than 50%) in GS activity was observed at the end of the experiment, indicating that GS was affected by this inhibitor. Similar experiments were performed using DBMIB (Fig. 5), which had no clear effect even after 24 h.

FIG. 5.

Effect of the addition of DCMU and DBMIB on the GS activity of Prochlorococcus strain PCC 9511. Samples were taken at the indicated times from cultures with no addition (control, ▪) or after 0.3 μM DCMU (●) or 0.06 μM DBMIB (▴) addition. Inhibitors were added at time zero. GS activity at time zero was 3.66 U · mg of chlorophyll⁻¹.

EIA determination of GS concentration.

It has been previously demonstrated that GS can be regulated at the level of enzyme concentration in other photosynthetic microorganisms, such as algae (9, 10), but it seems that GS concentration in cyanobacteria remains unchanged (19), regulation affecting only its activity and glnA expression. We have performed initial studies measuring the GS antigen concentration in Prochlorococcus samples by EIA in order to assess whether GS is regulated at this level. Table 2 shows the effects of different conditions on the GS protein concentration in Prochlorococcus strains PCC 9511 and SS120, as measured using antibodies raised against the GS of Synechocystis strain PCC 6803. The only clear change was observed after the addition of azaserine, which induced significant increases in the GS protein concentration in both strains. The effect of DBMIB on activity (48% reduction in SS120; data not shown) was not reflected in the enzyme concentration, which did not change.

TABLE 2.

GS protein concentrations in crude extracts of Prochlorococcus PCC 9511 and SS120^a

Condition	GS protein concn (relative area, arbitrary units)
	PCC 9511	SS120
Control (light, 400 μM NH4+)	100	100
N starvation	119	80
Darkness		81.6
0.3 μM DCMU	132	129
0.06 μM DBMIB	78	130
100 μM MSX	114	67
100 μM azaserine	167	268

^aSamples were taken after the cultures were grown for 24 h under the indicated conditions. Experimental procedures are described in Materials and Methods. 

DISCUSSION

Prochlorococcus is rapidly becoming a model organism because of its outstanding ecological success in oligotrophic areas of the oceans and its many unusual features (for a review, see reference 27). The U.S. Department of Energy has sequenced the genome of strain MED4, which is genetically very close, if not identical, to that of strain PCC 9511, which was used in the present study. In spite of this growing interest, basic knowledge of important metabolic pathways in Prochlorococcus is very limited. However, among the major remaining mysteries of this organism are the physiological mechanisms that it has developed to be able to proliferate in environments with extremely low concentrations of nutrients (27).

Here we have focused on the regulation in Prochlorococcus of GS—a pivotal enzyme in the nitrogen assimilatory pathway. Although most of the presented results are for strain PCC 9511, the same experiments were performed with strain SS120 (data not shown) as a representative of low-irradiance-adapted ecotypes. In all experiments, the results were similar to those obtained with PCC 9511, except for the effect of DBMIB, which provoked a 48% reduction in GS activity in SS120 after 24 h (data not shown). However, since SS120 is nonaxenic, this possible regulatory difference between high-light- and low-light-adapted ecotypes requires further confirmation after SS120 or another, equivalent strain is made axenic.

GS in eubacteria is a well-studied enzyme, constituting a classical and well-known example of complex regulatory cascades that inactivate the enzyme by adenylylation, depending on the carbon-nitrogen balance of the cells (34). Feedback inhibition by some amino acids and nucleotides has also been shown for cyanobacteria (5, 19, 23, 35). In addition, darkness (19) and ammonium (20) provoke the inactivation of GS in Synechococcus strain PCC 6301. A new kind of GS inactivation by protein-protein interactions that mediate the ammonium-induced inactivation of GS has been very recently observed in Synechocystis strain PCC 6803 (7); this inactivation is controlled by the NtcA system (8).

The first striking result of our studies is that GS activity is slightly decreased in Prochlorococcus strain PCC 9511 cells grown on ammonium and then transferred to PCR-S11 medium containing either nitrate, nitrite, or no nitrogen (Table 1). The standard response in most photosynthetic organisms (including cyanobacteria [20]) is an increase in enzymatic activity under conditions of nitrogen starvation. This unusual behavior could represent a specific adaptation of Prochlorococcus for colonizing very oligotrophic environments (27) in order to avoid the expensive production of GS protein when there is no nitrogen to assimilate. If continued N depletion occurs in nature, such a response could represent a selective advantage.

The fact that the studied Prochlorococcus strain did not grow on nitrate or nitrite (31; this work) is also a very uncommon and surprising situation in cyanobacteria, since in most cases both oxidized and reduced N sources can be assimilated through the pathway constituted by nitrate reductase, nitrite reductase, GS, and glutamate synthase (6). Our results (Table 1) suggest that transfer to nitrate or nitrite is in fact equivalent to nitrogen starvation in Prochlorococcus strain PCC 9511. A positive correlation has been shown between Prochlorococcus abundance and nitrate concentration in temperate areas (22), and nitrate addition was found to stimulate cell cycling of Prochlorococcus in the Mediterranean Sea in winter (42). Although nitrate could be reduced by coexistent bacteria prior to assimilation, as suggested by Rippka and coworkers (31), the possibility of nitrate assimilation in some Prochlorococcus strains remains open—in particular with regard to low-light-adapted ecotypes, which inhabit an environment not limited in nitrate, and in view of the genetic diversity (13, 33, 40) and wide distribution (26) of this cyanobacterium.

These results suggest that the studied Prochlorococcus strain (and probably ecotype) has experienced pressure inducing the loss of unnecessary genes that became useless in an environment where oxidized forms of nitrogen (nitrate or nitrite) were extremely scarce. Hence, the lack of genes encoding a nitrate-assimilating system in certain strains could save energy, leading to some evolutionary advantages and contributing to the reported compactness of the Prochlorococcus genome (36). This fact could be directly related to the very small size of Prochlorococcus cells, which has been proposed to be one of the critical factors in its ecological success, since its high surface area-to-volume ratio represents an advantage for the uptake of nutrients in oligotrophic areas of the oceans (27, 36).

Some recent reports have pointed to phosphorus limitation in some oceanic regions (41) where low chlorophyll concentrations are detected in spite of the abundance of nitrogen (2). The finding of a stronger effect on GS activity of P starvation than of N starvation (Fig. 2) was unexpected, since it can be assumed that the lack of nitrogen should mainly affect enzymes involved in nitrogen assimilation. This result could indicate that P limitation is more stressing to Prochlorococcus than the absence of N. The effect of P starvation on the Prochlorococcus cell cycle is much more marked than is that of N depletion, since prolonged P starvation provokes a block in all phases of the cell cycle rather than at a specific point (25). Furthermore, upon P addition, cells which are blocked in DNA synthesis cannot restart cycling and die. The presence of the pstS gene (expressed only under conditions of P depletion [27]), however, demonstrates that Prochlorococcus possesses mechanisms of adaptation to P limitation.

The effect of MSX on GS regulation has been studied with other unicellular organisms, such as cyanobacteria (24) and green algae (9). This inhibitor provokes rapid inactivation in Anabaena sp. strain PCC 7120 (24) at concentrations lower than those required for inactivation in vitro (1 μM MSX induces a drastic decrease in GS activity in a few hours in vivo, whereas 100 μM is necessary for in vitro inactivation). This result is probably due to transport mechanisms producing a very high intracellular concentration of MSX (3 mM). However, we observed that the addition of 1 mM MSX to cultures induced only partial inactivation in Prochlorococcus strain PCC 9511 (Fig. 3). This result could be the consequence of inefficient transport of MSX into these cells.

Inhibition of glutamate synthase had the opposite effect; GS activity (Fig. 3) and GS protein level (Table 2) increased quickly in ammonium-grown Prochlorococcus cultures after the addition of 100 μM azaserine. Very similar results were found for Synechocystis strain PCC 6803 after the addition of azaserine to ammonium-grown cultures (20). Still, the overall situation is different, since GS from Synechocystis grown on nitrate is inactivated by ammonium (20), so that the addition of azaserine induces reactivation of the enzyme. These results strongly suggest that (i) the glutamine concentration is not involved in the regulation of GS in Prochlorococcus, as occurs in other cyanobacteria (20) but not in other organisms (4, 9), and (ii) the mechanism inducing the upregulation of GS after the addition of azaserine is present in Prochlorococcus (as evidenced by the similar responses of Prochlorococcus strain PCC 9511 and Synechocystis strain PCC 6803), although other regulatory systems are clearly different.

The small change induced by darkness in GS activity from Prochlorococcus (Fig. 4) represents another main difference in the regulation of this enzyme compared to its regulation in most photosynthetic organisms, the usual situation being that darkness promotes a marked decrease in GS activity (10, 19, 32, 37, 38). Prochlorococcus can grow at a depth of 200 m, receiving less than 0.1% of the surface irradiance. The extremely low irradiance reaching this habitat has induced low-light-adapted ecotypes to develop a variety of adaptation mechanisms, including a high chlorophyll b/chlorophyll a ratio (28), large amounts of very efficient antenna proteins (15, 29), and the multiplication of the pcb genes encoding such antennae (12). Other modes of acclimation to low light could be expected for Prochlorococcus metabolic pathways directly related to photosynthesis, such as nitrogen assimilation.

Our results obtained using antagonistic inhibitors of photosynthetic electron transport show that the addition of DCMU induced a reduction in GS activity, while DBMIB provoked no clear change. Since both products block electron flow (30, 39), inducing the plastoquinone pool to be fully oxidized (DCMU; blocking between the Photosystem II complex and the plastoquinone pool) or fully reduced (DBMIB; blocking between the plastoquinone pool and the cytochrome b₆f complex), we propose that the redox state of the plastoquinone pool (and not the electron flow in a general sense) acts as a physiological sensor for GS regulation in Prochlorococcus.

In conclusion, we have found some evidence in our studies on GS regulation that suggests the occurrence in Prochlorococcus of adaptation mechanisms in the nitrogen assimilation pathway which may play a role in its ability to survive in conditions of strong nutrient limitation.