P_II-Regulated Arginine Synthesis Controls Accumulation of Cyanophycin in Synechocystis sp. Strain PCC 6803

Abstract

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-l-glutamate kinase, by signal transduction protein P_II.

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid) is a nitrogen-rich reserve polymer present in most cyanobacteria (reviewed in references 4, 5, 34, and 43) as well as in some heterotrophic bacteria (27, 49). It consists of a poly-α-aspartic acid backbone, with arginine linked to the β-carboxyl group of almost every aspartyl residue via isopeptide bonds (44). Cyanophycin is synthesized by a single enzyme, cyanophycin synthetase, from aspartate and arginine in an ATP-dependent reaction using a still-unidentified primer (1, 2, 8, 17, 42, 48). The amount of cyanophycin in cyanobacteria varies considerably with growth conditions. Its content is usually less than 1% of dry weight in rapidly growing cultures but is high (up to 18%) in stationary-phase cultures and under conditions of unbalanced growth such as sulfate or phosphate limitation (6, 30, 40, 45). When nitrogen-starved cyanobacterial cultures were provided with combined nitrogen sources, a rapid but transient accumulation of cyanophycin occurred (3). The cyanophycin contents of Anabaena cylindrica and Synechocystis sp. strain PCC 6803 increased severalfold when translation was inhibited by chloramphenicol (6, 41), indicating that rapid synthesis of the polymer did not depend on de novo synthesis of cyanophycin synthetase and that consumption of amino acids by protein synthesis may compete with the accumulation of cyanophycin. Furthermore, no correlation was found between the extractable activity of cyanophycin synthetase and the rate of polymer accumulation (31). These and several similar studies could not, so far, elucidate the mechanism(s) by which cyanophycin accumulation is regulated. Recently, it was shown that the genes for cyanophycin metabolism are under nitrogen control in the diazotrophic strain Anabaena sp. strain PCC 7120 (35). Furthermore, an involvement of the signal transduction protein P_II in the control of cyanophycin synthesis was suggested (19, 29) (see below).

The cyanobacterial P_II protein is a member of the large family of P_II signal transduction proteins, which play pervasive roles in nitrogen control in bacteria, plants, and some archaea (for recent reviews, see references 7 and 12). Similar to its Escherichia coli counterpart, P_II from the cyanobacterium Synechococcus elongatus PCC 7942 binds ATP and 2-oxoglutarate in a synergistic manner (13, 24). In the presence of increased 2-oxoglutarate levels, corresponding to nitrogen-limited conditions, P_II is phosphorylated at seryl residue 49 (14). Dephosphorylation of P_II-P in Synechocystis sp. strain PCC 6803 is catalyzed by PphA, a phosphatase of the PP2C family (23), under conditions of low 2-oxoglutarate levels (39). Recently, the first molecular target of P_II was identified in S. elongatus: N-acetyl-l-glutamate kinase (NAGK), which catalyzes the committed step in the cyclic arginine synthesis pathway (11). Its activity is strongly enhanced by complex formation with the nonphosphorylated form of P_II, signaling nitrogen-excess conditions (19). Furthermore, the effector molecules 2-oxoglutarate, ATP, and ADP as well as Ca²⁺ modulate NAGK-P_II complex formation (32). The NAGK-P_II interaction seems to be universally conserved in oxygenic phototrophs, including higher plants (10, 46). Based on the key function of NAGK in arginine synthesis, we hypothesized that P_II activation of arginine synthesis might play a role in the accumulation of cyanophycin under nitrogen-excess conditions (19). However, S. elongatus is one of a few cyanobacteria not able to synthesize cyanophycin, precluding investigation of this issue.

A mutant of the P_II phosphatase PphA homologue in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 displays increased P_II phosphorylation levels in heterocysts (28) and is impaired in formation of cyanophycin polar bodies. By contrast, cyanophycin accumulated in vegetative cells, implying that P_II might be involved in controlling cyanophycin distribution along the filament. However, further studies of P_II function in Anabaena are impeded by the lack of P_II-null mutants in the Nostocales group (18). The present study was conducted to clarify the role of P_II in cyanophycin accumulation. The strain Synechocystis PCC 6803 was used since it produces cyanophycin (1, 15) and mutants in the P_II signaling system are available. In particular, we used a P_II-null mutant (22) and a PphA-deficient mutant (23), which exhibits significantly delayed P_II dephosphorylation upon ammonium addition (25), to study the correlation between P_II phosphorylation status, NAGK activity, and cyanophycin accumulation following nitrogen-excess treatments.

The transformable wild-type Synechocystis sp. strain PCC 6803 (15) and the isogenic Synechocystis mutants MPphA (PphA deficient; pphA::kan [23]) and ΔP_II (P_II deficient; glnB::spc [22]) were routinely grown in liquid BG11 medium (38) supplemented with 5 mM NaHCO₃. The MPphA strain was maintained with kanamycin (50 μg ml⁻¹) selection and the ΔP_II-strain with spectinomycin selection (35 μg ml⁻¹). In the first set of experiments, wild-type cells of Synechocystis sp. strain PCC 6803 and the mutants ΔP_II and MPphA were shifted from nitrogen-poor to nitrogen-excess conditions. Nitrogen-limited cultures were prepared by harvesting cells from 2 ml of nitrate-replete stock culture and resuspending them in 100 ml of modified BG11 medium (low-N BG11) containing 1 mM of nitrate. These cultures were grown in triple-baffled flasks with shaking at 30°C, under continuous illumination of 50 μmol photons m⁻² s⁻¹ from white fluorescent lamps. When an optical density at 750 nm of 0.8 to 1.0 was reached (after approximately 4 days for the wild-type and MPphA strains and 5 days for the ΔP_II strain), cells started to get slightly bleached due to consumption of nitrate. After the time zero aliquots were taken ammonium chloride was added to a final concentration of 5 mM (nitrogen excess), and aliquots of the culture were harvested in the course of time. From these samples, the accumulation of cyanophycin, activity of NAGK, and phosphorylation status of P_II were analyzed. Furthermore, at selected time points, the amount of cellular arginine was determined. Cyanophycin was extracted (40) from 10-ml samples and enzymatically hydrolyzed by recombinant cyanophycinase (37) and recombinant isoaspartyl dipeptidase from Synechocystis sp. strain PCC 6803 (20) to arginine and aspartic acid. The mass of the polymer was calculated from the liberated aspartic acid, quantified enzymatically (33). Values were reproducible within ±5%. For the determination of NAGK activity and the phosphorylation status of P_II, cell extracts of the samples were prepared using a RiboLyser (Hybaid) as described previously (19) and protein concentration was estimated using the Bradford assay (9). One hundred micrograms of extract protein was used for a NAGK assay, and 5 μg of protein was used for P_II phosphorylation state analysis. To measure the intracellular arginine level, cells from 4 ml of culture were harvested, suspended in 1 ml of 80% ethanol, and incubated for 3 h at 65°C. Following centrifugation, the supernatant was dried and the arginine content was determined by high-pressure liquid chromatography according to reference 16.

As shown in Fig. 1A, wild-type cells rapidly accumulated cyanophycin following ammonium treatment. By contrast, cyanophycin accumulation was completely absent in the P_II-deficient mutant. The PphA-deficient strain showed an intermediate phenotype, having a delayed accumulation of cyanophycin compared to the wild type. Determination of NAGK activity revealed that, following ammonium upshift, wild-type cells rapidly increased the activity of this enzyme. By contrast, the P_II-deficient mutant was unable to increase NAGK activity, the same result observed previously in P_II-deficient cells of S. elongatus sp. strain PCC 7942 (19). The PphA-deficient cells showed a delayed increase of NAGK activity, compared to the wild type. Quantification of intracellular arginine following ammonium upshift revealed that the arginine level in wild-type cells increased immediately upon N upshift whereas it remained low in the P_II-deficient mutant (Fig. 2). In the PphA mutant, increased arginine levels could be observed only in the last sample, which showed accumulated cyanophycin.

FIG. 1.

Analysis of cyanophycin accumulation (A) and NAGK activity (B) in cell extracts prepared from Synechocystis sp. strain PCC 6803 cells, which were grown under nitrate-limiting conditions and shifted to 5 mM ammonium chloride. Immediately before the shift (time zero) and after different time points, as indicated, aliquots were removed and extracts were prepared. The results from the Synechocystis wild-type strain (black bars), the MPphA strain (gray bars), and the ΔP_II strain (open bars) are shown. Note that no cyanophycin was detectable in ΔP_II cells. (C) Determination of the phosphorylation state of P_II in the extracts of Synechocystis wild-type (top) and MPphA (bottom) cells that were analyzed as for panels A and B. The phosphorylation status of P_II is shown on the left, with the superscripts indicating the numbers of phosphorylated subunits.

FIG. 2.

Quantification of arginine in ethanolic extracts from Synechocystis sp. strain PCC 6803 cells which had been grown under nitrate-limiting conditions and shifted to 5 mM ammonium chloride at time zero. The concentration of arginine is given as pmol Arg extracted per unit of optical density at 750 nm (OD₇₅₀) of cells. The results from the Synechocystis wild-type strain (black bars), the MPphA strain (gray bars), and the ΔP_II strain (open bars) are shown.

The activation state of NAGK, cyanophycin accumulation, and intracellular arginine concentration strongly correlated with the phosphorylation state of P_II (Fig. 1C). Previously, we demonstrated that, in Synechococcus sp. strain PCC 7942, the nonphosphorylated form of P_II strongly activates NAGK activity (19). Similarly, in the Synechocystis sp. strain PCC 6803 wild-type cells, dephosphorylation of P_II correlates with an increase in NAGK activity. The increased NAGK activity is accompanied by increased intracellular arginine and cyanophycin concentrations. In contrast, the delay in P_II dephosphorylation in the MPphA strain correlated with delayed NAGK activation and cyanophycin and arginine accumulation. Immunoblot analysis using NAGK-specific antibodies revealed that the amount of NAGK protein did not significantly change during the time course of the experiment (data not shown).

The above results strongly suggested that P_II-mediated NAGK activation is responsible for increased arginine synthesis, which then leads to cyanophycin accumulation. To verify independently that impaired cyanophycin synthesis in P_II-deficient cells is indeed due to limiting arginine levels and not caused by impaired cyanophycin synthetase activity (26), ammonium upshift experiments in the presence of 5 mM arginine were carried out with wild-type and P_II-deficient cells (Fig. 3). Synechocystis has a highly active arginine transport system (28), resulting in a rapid uptake of externally added arginine. As shown in Fig. 3A, the P_II-deficient mutant, despite low NAGK activity, was now able to accumulate cyanophycin, although to a lesser extent than the wild type (Fig. 3B). The difference between wild type and mutant may be due to the lack of internally synthesized arginine in the ΔP_II strain or may indicate an additional requirement for P_II in cyanophycin synthesis. The recently discovered P_II receptor PamA in Synechocystis may be considered in this context (47). In any case, cyanophycin accumulation can be restored in the P_II-deficient mutant by bypassing the impaired NAGK activity through external addition of arginine, implying that cyanophycin synthesis in the ΔP_II strain was limited by the availability of arginine.

FIG. 3.

Analysis of cyanophycin accumulation (A) and NAGK activity (B) in cell extracts from Synechocystis sp. strain PCC 6803 cells, which had been grown under nitrate-limiting conditions and shifted to 5 mM ammonium chloride with 5 mM arginine. Immediately before the shift (time zero) and after different time points, as indicated, aliquots were removed and extracts were prepared. The results from the Synechocystis wild-type strain are shown by the black bars and those from the ΔP_II strain by open bars.

Arginine has a dual role in cyanobacteria, first as an amino acid for protein synthesis and second as a nitrogen buffer, storing excess nitrogen in the form of cyanophycin and making it easily available through efficient arginine metabolism (21, 36). P_II controls the committed step in arginine synthesis as it activates NAGK activity by complex formation. In addition to increasing NAGK catalytic activity, complex formation with P_II also causes a dramatic reduction in arginine feedback inhibition. Whereas free NAGK was almost completely inhibited by arginine concentrations above 50 μM, the P_II-complexed NAGK was barely inhibited (32). Therefore, under physiological conditions of P_II-NAGK complex formation, efficient arginine synthesis occurs in the presence of appreciably higher levels of arginine compared to conditions favoring P_II-NAGK complex dissociation. Complex formation occurs with nonphosphorylated P_II at low levels of 2-oxoglutarate, corresponding to nitrogen-rich conditions. Under nitrogen-poor conditions, however, complex formation is impaired, since P_II is phosphorylated and the 2-oxoglutarate concentrations are high (19, 32). Cyanophycin synthetase in Synechocystis sp. strain PCC 6803 has a K_m for arginine of 49 μM (2), a concentration which is already inhibitory for free NAGK but not for NAGK in complex with P_II. Therefore, under nitrogen-poor conditions, cyanophycin cannot be formed. Instead, arginine levels should be just sufficient to meet the requirement for protein synthesis, since the K_m values of aminoacyl-tRNA synthetases for their cognate amino acids are usually in the micromolar range. Under conditions of nitrogen excess, however, the NAGK-P_II complex is formed and arginine synthesis is stimulated, allowing cyanophycin synthesis to occur. The other substrates of cyanophycin synthetase, aspartate and ATP (exhibiting K_m values for Asp of 0.45 mM and for ATP of 0.2 mM [2]), do not seem to limit the reaction in the P_II-deficient mutant, since arginine addition alone was sufficient to restore cyanophycin synthesis. The regulation of NAGK activity by P_II in response to the nitrogen status thus provides the mechanistic basis for the dual role of arginine: in the nonactivated state, NAGK activity is sufficient to provide arginine for the purpose of protein synthesis; in the P_II-activated state, excess nitrogen can be stored in the form of cyanophycin.