Signal Transduction Protein PII Phosphatase PphA Is Required for ...

JOURNAL OF BACTERIOLOGY, Oct. 2005, p. 6683–6690 0021-9193/05/$08.00⫹0 doi:10.1128/JB.187.19.6683–6690.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 187, No. 19

Signal Transduction Protein PII Phosphatase PphA Is Required for Light-Dependent Control of Nitrate Utilization in Synechocystis sp. Strain PCC 6803 Nicole Kloft and Karl Forchhammer* Institut fu ¨r Mikrobiologie und Molekularbiologie, Justus-Liebig-Universita ¨t Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany Received 18 May 2005/Accepted 18 July 2005

Signal transduction protein PII is dephosphorylated in Synechocystis sp. strain PCC 6803 by protein phosphatase PphA. To determine the impact of PphA-mediated PII dephosphorylation on physiology, the phenotype of a PphA-deficient mutant was analyzed. Mutants lacking either PphA or PII were impaired in efficient utilization of nitrate as the nitrogen source. Under conditions of limiting photosystem I (PSI)-reduced ferredoxin, excess reduction of nitrate along with impaired reduction of nitrite occurred in PII signaling mutants, resulting in excretion of nitrite to the medium. This effect could be reversed by increasing the level of PSI-reduced ferredoxin. We present evidence that nonphosphorylated PII controls the utilization of nitrate in response to low light intensity by tuning down nitrate uptake to meet the actual reduction capacity. This control mechanism can be bypassed by exposing cells to excess levels of nitrate. Uncontrolled nitrate uptake leads to light-dependent nitrite excretion even in wild-type cells, confirming that nitrate uptake controls nitrate utilization in response to limiting photon flux densities. NtcA-activated gene expression (5). A central molecule for perception and signaling of the cellular nitrogen status in bacteria is the PII signal transduction protein (2, 7, 37). The PII protein family is one of the most widely distributed families of signal transduction proteins, whose members are present in all domains of life (2). PII proteins play ubiquitous roles in various aspects of nitrogen regulation, and they display a remarkable functional diversity with respect to the targets of regulation (2, 7, 37). The three-dimensional structure of various PII proteins is highly conserved, and in all cases analyzed so far, the PII proteins recognize adenylate nucleotides and 2-oxoglutarate (9, 24, 30, 46), which control the reactivity of PII towards various targets (23, 31). Furthermore, PII proteins may be covalently modified at a surface-exposed loop, the so-called “T-loop” (named after the uridylylated tyrosyl residue 51 at the tip of the loop in PII proteins from proteobacteria), which further modulates signal output by PII. Uridylylation of Y51 in proteobacteria is reversibly regulated by the cellular glutamine level, which serves as the primary nitrogen signal in these organisms (for a review, see reference 37). PII is modified by phosphorylation at seryl residue 49 in response to the cellular nitrogen and carbon supply, as shown for the freshwater strains Synechococcus elongatus and Synechocystis sp. strain PCC 6803. In cells grown in ammonium, PII is almost completely nonphosphorylated. In nitrate-supplemented cells, intermediate levels of PII phosphorylation, which are modulated by the inorganic carbon supply to the cells, are observed, and the highest levels of PII phosphorylation occur under conditions of nitrogen starvation (7, 12). Targets of PII signaling are beginning to emerge. Previously, it was shown in a PII-deficient mutant of Synechococcus sp. strain PCC 7942 that nitrate utilization was no longer regulated by the carbon supply to the cells. Furthermore, inhibition of NRT activity by ammonium was also impaired (12, 19, 29), suggesting that a

Cyanobacteria are oxyphototrophic bacteria capable of growing solely by using inorganic nutrients in light and using water as an abundant source of reductant for assimilatory processes. For nondiazotrophic freshwater cyanobacteria, such as Synechocystis sp. strain PCC 6803, nitrate is probably the most abundant source of combined nitrogen (14). Nitrate utilization requires uptake through an ATP-binding cassette-type transporter (NRT), which is a nitrate-nitrite bispecific transporter encoded by the nrtABCD genes (32, 33, 39, 40). Intracellular nitrate is reduced to nitrite by nitrate reductase (NR) and subsequently to ammonium by nitrite reductase (NiR). Ammonium is then assimilated into organic material via the glutamine synthetase-glutamate synthase cycle (4). Both NR and NiR use photosystem I (PSI)-reduced ferredoxin as their physiological electron donor (35). The reduction of nitrate requires two electrons, whereas six electrons are needed for nitrite reduction. Under photoautotrophic growth conditions, reduction of nitrate and reduction of nitrite are coupled to photosynthetic oxygen evolution in vivo and thus can be considered genuine photosynthetic processes (4). Utilization of nitrate in cyanobacteria is subject to global nitrogen control (4, 16), a process in which ammonium, through assimilation by the glutamine synthetase-glutamate synthase pathway, depresses the utilization of alternative nitrogen sources. Global nitrogen control operates both at the level of enzyme activity and at the level of gene expression, mediated by the transcriptional regulator NtcA. Addition of ammonium to cells growing in the presence of nitrate results in an immediate inhibition of NRT activity and depression of * Corresponding author. Mailing address: Institut fu ¨r Mikrobiologie und Molekularbiologie, Justus-Liebig-Universita¨t Giessen, HeinrichBuff-Ring 26-32, D-35392 Giessen, Germany. Phone: 49 641-9935545. Fax: 49 641-9935549. E-mail: [email protected] .de. 6683

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component of NRT was regulated by PII. Studies with S49 mutants of PII, which potentially mimic the phosphorylated protein, showed that NRT was regulated by PII even without changing the PII modification status (28). A recent study confirmed that the phosphorylation status of PII does not affect the regulation of nitrate uptake in response to ammonium in Synechocystis sp. strain PCC 6803 (27). Studies by Hisbergues et al. (19) suggested that in Synechocystis sp. strain PCC 6803, highaffinity bicarbonate uptake is also regulated by PII without requiring PII modification. Moreover, NtcA-activated gene expression under conditions of nitrogen starvation was shown to depend on PII signaling (1, 41). In these cases, the direct targets of interaction with PII are not yet known at the molecular level. Recently, N-acetyl-L-glutamate kinase (NAGK) was identified as the first molecular target of PII signaling in a cyanobacterium (3, 15). NAGK catalyzes the first committed step in arginine biosynthesis and forms a tight complex with nonphosphorylated PII. Binding of PII strongly enhances the catalytic activity of this enzyme, whereas no NAGK activation or complex formation occurs with S49-modified PII (15, 34). The cellular signal for PII phosphorylation is an elevated level of 2-oxoglutarate (11, 21), which serves as a signaling molecule of the cellular carbon/nitrogen balance (8, 36). Phosphorylated PII protein (PII-P) is dephosphorylated by a type 2C protein phosphatase, termed PphA, which was discovered in Synechocystis sp. strain PCC 6803 (22). In vitro analysis with purified components revealed that in the presence of ATP, dephosphorylation of PII-P by PphA responded in a highly sensitive manner to subtle changes of 2-oxoglutarate in the submillimolar concentration range and to a lesser extent also to oxaloacetate (7, 44). Elevated levels of these effector molecules lead to inhibition of PphA-mediated PII-P dephosphorylation. A PphA-deficient mutant was unable to rapidly dephosphorylate PII-P in response to various signals (7, 22), in agreement with the finding that PII-P appears to be a very poor substrate for other cellular phosphatases (26). The abundance of PphA was shown to increase in response to elevated nitrate/ nitrite levels, suggesting that this enzyme has an important function under these conditions (26). Detailed analysis of the phenotype of a PphA-deficient mutant should provide insights into the function(s) of the PII phosphorylation/dephosphorylation cycle in this cyanobacterium. Here, we describe a new regulatory mechanism in which PII-P dephosphorylation by PphA is required to fine-tune nitrate uptake under conditions of limiting PSI-reduced ferredoxin to prevent the formation of excess nitrite.

J. BACTERIOL. centrifugation and washed with the appropriate medium (see below) to remove the antibiotic from the MPphA culture. The strains were mixed and grown in nitrate-, ammonium-, or urea-limited medium (final concentration, 0.5 mM) at an illumination of 40 ␮mol photons s⫺1 m⫺2. When the nitrogen source was exhausted (as deduced from growth arrest and onset of chlorosis), an aliquot of the culture was diluted (1:5) into fresh medium containing limiting amounts of the nitrogen source. This procedure was repeated three times. Appropriate dilutions of liquid cultures were plated on BG11N plates with and without kanamycin (solidified by the addition of 0.9% [wt/vol] of Gel-Rite [Roth]) to determine the CFU of MPphA (revealed by CFU on kanamycin plates) and wild-type cells (difference of CFU between nonselective and kanamycin plates). The plates were incubated at 30°C with a PPFD of 30 ␮mol photons s⫺1 m⫺2. Control experiments revealed that during the time course of the experiment, omission of the selective antibiotic did not lead to a loss of the mutation. DNA isolation and Southern blot analysis. Extraction of chromosomal DNA from cultures of the competition experiment was performed by using the QIAGEN DNeasy tissue kit. A 1.5-␮g sample of SmaI-restricted DNA was applied to each lane of a 1% (wt/vol) agarose gel. Electrophoretic conditions, transfer of DNA to a nylon membrane (Roti-Nylon plus, Roth), and hybridization conditions were according to standard protocols (45). The pphA gene probe used in DNA-DNA hybridization, a 0.46-kb DNA fragment corresponding to nucleotides 317 to 765 of the pphA (sll1771) coding region (25), was generated by restriction of the expression plasmid pT7-7pphA (20, 22) with KpnI and SmaI. This DNA probe was labeled with [32P]dCTP by using the Megaprime DNA labeling kit (Amersham Pharmacia). The hybridization signals were visualized by exposing the membrane to a phosphorimager screen (K-Imaging screen, Bio-Rad, Hercules, CA), which was recorded in a phosphorimager (Molecular Imager FX, Bio-Rad). Quantification was performed using the Bio-Rad Quantity One software. Determination of nitrate uptake and nitrite excretion. To determine nitrate uptake and nitrite excretion, cells grown in BG11N medium were harvested by centrifugation, washed with BG11 medium without any nitrogen source, and resuspended in the same medium to an OD750 of 1. The assays were started by addition of 200 ␮M NaNO3 to the cell suspension. To examine the effect of ammonium, NH4Cl (2 mM final concentration) was added. The culture was incubated under light and with shaking. Nitrate uptake was determined by estimating the concentration of nitrate in 1-ml aliquots of the medium. Therefore, the cells were removed from the medium by centrifugation, and the absorbance of nitrate was measured at 210 nm. Since the absorbance at 210 nm detects both nitrate and nitrite, the apparent nitrate values were corrected for the presence of nitrite. To determine the nitrite excretion, the nitrite concentration in aliquots of the medium was quantified by colorimetric assay (47). To analyze nitrite utilization in more detail, precultures of the wild type, MPphA, and ⌬PII were grown in modified BG11 medium containing nitrite (5 mM final concentration), in which molybdate was replaced by tungstate (4.8 ␮M). At the mid-exponential phase of growth, the cells were harvested by centrifugation and washed in combined nitrogen-free medium (BG110). The cells were resuspended in modified BG110 medium (containing tungstate) to an OD750 of approximately 1, and NaNO2 and/or NaNO3 (each 100 ␮M) was added. Determination of the modification state of PII. The phosphorylation state of PII in vivo was analyzed by nondenaturing polyacrylamide gel electrophoresis followed by immunoblot analysis of PII as described previously (10). PII0, PII1, PII2, and PII3 represent isoforms of the trimeric PII carrying no, one, two, and three phosphate groups, respectively. Enzyme assays. Determination of nitrate reductase (17) and nitrite reductase (18) was performed in mixed alkyltrimethylammonium bromide (MTAB)-permeabilized cells with dithionite-reduced methyl viologen as the reductant.

MATERIALS AND METHODS Strains and growth conditions. Synechocystis sp. strain 6803T (13), the derived PphA-deficient mutant MPphA (pphA::kan) (22), and the PII null mutant ⌬PII (glnB::spec) (19) were grown in liquid BG11 medium (42) supplemented with 5 mM NaHCO3 and 17.6 mM NaNO3 (BG11N) as the nitrogen source. The cultures were incubated in baffled Erlenmeyer flasks capped with silicone sponge closures (Bellco Glass, Vineland, NJ) and rotated with 150 rpm for efficient gas transfer. Cells were grown under photoautotrophic growth conditions at 25°C at a photosynthetic photon flux density (PPFD) of 40 ␮mol photons s⫺1 m⫺2 from white fluorescent tubes (LUMILUX de Luxe Daylight, Osram). The mutant MPphA was maintained with kanamycin (30 ␮g ml⫺1) and the ⌬PII mutant with spectinomycin (35 ␮g ml⫺1). Growth of the cultures was monitored by determination of the optical density at 750 nm (OD750). For competition experiments, cultures of Synechocystis sp. strain PCC 6803 and the mutant MPphA, both with identical ODs (0.2), were harvested by

RESULTS Growth phenotype of a PphA-deficient mutant. Previous investigations showed that PphA is required for the rapid dephosphorylation of PII-P by various treatments, such as addition of ammonium, depletion of inorganic carbon, or inhibition of photosynthetic electron flow (7, 22). Despite impaired dephosphorylation of PII, PphA-deficient mutants retained their capacity to acclimate rapidly to the addition of ammonium, as revealed by glutamine synthetase assays (26), and their growth, as deduced from growth rates, was not obviously impaired with ammonium as the nitrogen source (22). This imposed the ques-

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PII CONTROL OF NITRATE UTILIZATION IN SYNECHOCYSTIS

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FIG. 1. Growth competition experiment between wild-type and PphA-deficient Synechocystis sp. strain PCC 6803 cells. (A) Ratio of MPphA cells to total cells in mixed cultures during growth competition experiments using nitrate (black bars), ammonium (gray bars), or urea (white bars) as nitrogen sources (for experimental details, see Materials and Methods). Samples were removed at the beginning of the experiments (1), before the second transfer to fresh medium (2), immediately after the third transfer (except for the urea experiment) (3), and 2 days after the third transfer (4). (B) Restriction map of the pphA region in the chromosomal DNA of wild-type Synechocystis sp. strain PCC 6803 (above) and the PphA-deficient mutant (below) in which the gene is interrupted by the kanamycin resistance cartridge aphI (23). (C) Southern blot analysis of chromosomal DNA prepared from a mixed culture of wild-type and PphA-deficient Synechocystis sp. strain PCC 6803 cells competing for nitrate as the nitrogen source. Total DNA was prepared from the cells, restricted with SmaI, and hybridized with a KpnI-SmaI DNA fragment (see panel A) as described in Materials and Methods. As controls, the restriction patterns of DNA from wild-type (WT) and MPphA cells are shown as indicated. Lane numbers correspond to the sampling points detailed in panel A.

tion of the physiological significance of PphA-mediated dephosphorylation of PII-P. To answer this, we examined the growth phenotype of PphA-deficient mutants (MPphA) in more detail by performing growth competition experiments in mixed cultures (see Materials and Methods). When the cells were competing for limiting amounts of nitrate, wild-type cells outgrew PphA-deficient mutants. After three repetitions of dilution and growth, only approximately 10% of the CFU were derived from the MPphA cells (Fig. 1A). By contrast, when the experiment was performed with ammonium- or urea-limited medium the proportion of wild-type and mutant cells did not change. In accord with plating analysis, Southern blot analysis of isolated chromosomal DNA from samples of the nitratelimited mixed culture revealed the gradual disappearance of the PphA mutant while the relative proportion of wild-type cells increased (Fig. 1B and C). Nitrate utilization and nitrite excretion in mutants of the PII signaling system. The competitive disadvantage of MPphA cells relative to wild-type cells in nitrate-supplemented medium suggested a subtle impairment in nitrate utilization. Previous analysis of PII-deficient mutants of Synechococcus sp. strain PCC 7942 revealed the accumulation of nitrite during nitrate-supplemented growth (12). Determination of nitrite in

the culture medium of MPphA cells indeed showed increased formation of nitrite compared to wild-type cells. In standard BG11 medium (with a final concentration of 17.6 mM nitrate), six times more nitrite was detected in MPphA cultures in the exponential phase of growth than in wild-type cultures (Fig. 2). To analyze the rate of nitrate consumption and concomitant formation of nitrite in vivo, cells were incubated in medium containing 200 ␮M nitrate, and at different times, nitrate depletion and nitrite accumulation were analyzed. As shown in Fig. 3A, MPphA cells took up nitrate even more rapidly than the wild type but excreted the excess of reduced nitrate back to the medium in the form of nitrite. Addition of ammonium resulted in a stop of nitrate uptake in both wild-type and MPphA cells (Fig. 3B), and as a consequence, MPphA cells produced almost no nitrite. Further experiments demonstrated that the responses of NRT activity toward various ammonium concentrations were identical in wild-type and MPphA cells (data not shown). In the PII-deficient strain (⌬PII), however, nitrate consumption was not affected by the presence of ammonium (Fig. 3B, triangles). Part of the consumed nitrate was reexcreted to the medium as nitrite, confirming previous reports that the PII protein is required for the control of nitrate utilization in response to a short-term exposure to ammonium

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J. BACTERIOL. TABLE 1. Light-dependent nitrate consumption and nitrite formation (nmol min⫺1 mg chlorophyll a⫺1) by cells of wild-type Synechocystis sp. strain PCC 6803, the PphA-deficient mutant MPphA, and the PII-deficient mutant ⌬PII Nitrate consumption or nitrite formation by: PPFDa

WT NO3

10 40 120 a

FIG. 2. Nitrite formation by PphA-deficient cells in BG11N medium. Wild-type Synechocystis sp. strain PCC 6803 (squares) and MPphA (diamonds) cells were grown in BG11N medium to the midexponential phase of growth, harvested by centrifugation, washed twice in BG11N, and resuspended in BG11N to an OD750 of 1. Cells were incubated under standard growth conditions, and after different times, aliquots were removed and nitrite in the supernatant was determined.

(19, 27, 29), albeit by a mechanism that does not require PII dephosphorylation. Excretion of nitrite by MPphA cells indicates an imbalance of nitrate reduction compared to nitrite reduction. Therefore, the activities of nitrate and nitrite reductases (NR and NiR,

FIG. 3. (A) Consumption of 200 ␮M nitrate (filled symbols) and formation of nitrite (open symbols) by wild-type Synechocystis sp. strain PCC 6803 (squares) and PphA-deficient mutant MPphA (diamonds) cells. (B) Consumption of 200 ␮M nitrate (filled symbols) in the presence of 2 mM NH4Cl and formation of nitrite (open symbols) by cells of wild-type Synechocystis sp. strain PCC 6803 (squares), the PphA-deficient mutant MPphA (diamonds), and the PII-deficient mutant ⌬PII (triangles). The experiments were performed at a PPFD of approximately 35 ␮mol photons s⫺1 m⫺2. A representative of three independent experiments is shown.

⫺

118 ⫾ 2.5 203 ⫾ 10 296 ⫾ 1.2

⌬PII

MPphA NO2

⫺

2.5 ⫾ 1.2 7.5 ⫾ 3.7 2.5 ⫾ 3.7

NO3

⫺

137 ⫾ 12.5 250 ⫾ 5.0 340 ⫾ 1.2

NO2

⫺

45 ⫾ 5.0 34 ⫾ 1.2 10 ⫾ 0.0

NO3

⫺

NO2⫺

215 ⫾ 16 94 ⫾ 10 297 ⫾ 22 72 ⫾ 4.0 509 ⫾ 15 0 ⫾ 0

In ␮mol photons s⫺1 m⫺2.

respectively) were measured in MTAB-permeabilized cells, using methyl viologen as the electron donor. The activities of NR and NiR were not significantly different in nitrate-grown wildtype and MPphA cells. Wild-type cells exhibited activities for NR and NiR of 291 ⫾ 6 and 122 ⫾ 13 (activities are given in nmol substrate minute⫺1 mg chlorophyll a⫺1), respectively, compared to 284 ⫾ 24 and 101 ⫾ 6 in MPphA cells (the chlorophyll a contents of 1-ml cell suspensions at an OD750 of 1 corresponded to 5.5 ␮g for the wild type and 5.0 ␮g for MPphA cells). This argued against the possibility that the PphA mutation affects the levels of NR and NiR and thereby causes the excretion of nitrite. Furthermore, the intracellular concentrations of nitrite, determined in nitrate-grown cells, were almost identical in wild-type and PphA-deficient cells, indicating that differential accumulation of nitrite cannot account for the observed phenotype (data not shown). Light and reductant dependence of nitrite excretion. In the search for factors that were involved in nitrite excretion by MPphA cells, we observed that the extent of nitrite production strongly depended on the illumination conditions. To investigate the relation between PPFD and nitrite formation systematically, cultures of the wild type and the mutants MPphA and ⌬PII were incubated in medium containing 200 ␮M NaNO3 at PPFDs of 10, 40, and 120 ␮mol photons s⫺1 m⫺2. From the slopes of nitrite formation or nitrate removal (compare Fig. 3), the values shown in Table 1 were derived. Under low-light conditions, both mutants utilized more nitrate than the wild type and produced the largest amounts of nitrite. Thirty-three and 43% of the consumed nitrate was converted to nitrite and was excreted to the medium by MPphA and ⌬PII cells, respectively. As expected from the fact that nitrate utilization consumes PSI-reduced ferredoxin in cyanobacteria, elevated illumination increased nitrate consumption in all strains. Concomitantly with the increased PPFD, nitrite formation declined in both mutants. At an illumination of 40 ␮mol photons s⫺1 m⫺2, MPphA converted only 13% and ⌬PII 24% of the consumed nitrate to nitrite that was excreted to the medium. Under high-light conditions, nitrite production in all strains was barely detectable. To analyze the phosphorylation status of PII under the different light conditions, samples of wild-type and MPphA cultures were removed after 30 min of incubation (Fig. 4). Under low-light conditions (10 ␮E), an intermediate level of PII phosphorylation was observed, with appreciable levels of the nonphosphorylated form (PII0) and predominantly the forms with one and two phosphorylated subunits (PII1 and PII2). At higher light intensity, the phosphorylation status was


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FIG. 4. Photon flux density-dependent phosphorylation state of the PII protein in wild-type and PphA mutant (MPphA) cells of Synechocystis sp. strain PCC 6803. The cultures were treated in the same manner as for the experiment shown in Table 1 (see Materials and Methods). Immediately at the onset of the experiments, following the addition of nitrate to washed cells, the first samples were collected (c). After 30 min of incubation in the presence of different photon flux densities, as indicated (PPFD in ␮mol photons s⫺1 m⫺2), samples were collected and the phosphorylation state of PII was analyzed.

clearly increased, with only traces of unmodified PII and high levels of fully phosphorylated PII (PII3). By contrast, in MPphA cells, PII protein was present in its highly phosphorylated forms (PII2 and PII3) with no unmodified isoform detectable under any light conditions. Under low-light conditions, PSI-reduced ferredoxin, the electron donor for NR and NiR, is the limiting factor for the activities of these enzymes. Therefore, deficiency of electrons may cause the observed deregulation of nitrate/nitrite reduction, in spite of the identical activity levels of these enzymes as measured with artificial electron donors (see above). If this was the case, donation of electrons under light-limiting conditions should decrease nitrite formation in MPphA and ⌬PII. This possibility was investigated by addition of glucose, which can be utilized as a source of reductant in this facultatively photomixotrophic organism. One half of each culture was incubated in medium containing 200 ␮M NaNO3, and the other half was incubated in medium containing 200 ␮M NaNO3 together with 0.1% glucose at an illumination of 10 ␮mol photons s⫺1 m⫺2 (Table 2). In the presence of glucose, both mutants took up nitrate more rapidly and excreted less nitrite, such that the ratio of nitrite production per nitrate consumption decreased by a factor of two. The phosphorylation state of PII under those conditions is shown in Fig. 5. In the absence of glucose, three phosphorylated PII isoforms were observed in wild-type cells, the unmodified form (PII0) and the phosphorylated forms of PII with one and two phosphorylated subunits (PII1 and PII2), whereas only highly phosphorylated PII forms (PII2 and PII3) were observed in MPphA. The addition of glucose resulted in an increased phosphorylation of PII, with PII0 disappearing in cells of the wild type. TABLE 2. Nitrate consumption and nitrite formation (nmol min⫺1 mg chla⫺1) by cells of wild-type Synechocystis sp. strain PCC 6803, the PphA-deficient mutant MPphA, and the PII-deficient mutant ⌬PII in the presence or absence of 0.1% glucose Cell type

WT MPphA ⌬PII

NO3⫺ consumption

NO2⫺ formation

NO2⫺/NO3⫺ ratio (%)

⫹ Glucose ⫺ Glucose ⫹ Glucose ⫺ Glucose ⫹ Glucose ⫺ Glucose

220 ⫾ 29 204 ⫾ 32 227 ⫾ 47

104 ⫾ 9 139 ⫾ 4 155 ⫾ 4

9⫾5 41 ⫾ 5 45 ⫾ 4

1⫾1 52 ⫾ 9 58 ⫾ 5

4.1 20 20

1 37 37

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FIG. 5. Phosphorylation state of the PII protein in wild-type and PphA mutant (MPphA) cells of Synechocystis sp. strain PCC 6803 in the presence or absence of 0.1% glucose. Cultures were treated as for the experiment shown in Table 2. Samples were collected immediately at the onset of the experiment (0) and again after 0.5 h and 1 h, as indicated.

In vivo competition of nitrate and nitrite reductase. To elucidate whether the excretion of nitrite at 10 ␮mol photons s⫺1 m⫺2 in PII signaling mutants was caused by an impaired in vivo nitrite reductase activity, the utilization of nitrite in the absence or presence of nitrate was analyzed in a time course experiment. Both strains, the wild type and MPphA, exhibited almost identical consumptions of nitrite (Fig. 6A), revealing that in vivo nitrite reductase activity was not impaired in MPphA as long as nitrate was absent. In the presence of nitrate and nitrite at 100 ␮M (Fig. 6B), wild-type cells were able to utilize nitrite concomitantly with the utilization of nitrate. By contrast, MPphA cells utilized only nitrate, whereas the utilization of nitrite was impaired. The same result was obtained for the ⌬PII strain (data not shown). To clarify whether the activity of NR affects nitrite utilization in the mutants or whether nitrate had other effects, NR was poisoned by growing the cells in a medium in which molybdate was replaced by tungstate. Under these conditions, the cells expressed a nonactive nitrate reductase (18). Using such NR-poisoned cells, no difference in nitrite reduction could be detected between the wild type and the PII signaling mutant in the presence of nitrate (Fig. 6C). This confirmed that the impaired in vivo nitrite reduction in the mutants was indeed due to the competing activity of NR under conditions of limited PSI-reduced ferredoxin. Furthermore, these results implied that wild-type cells are able to balance the reduction of nitrate and nitrite by controlling the amount of nitrate reduction in response to the availability of reductant, a process that is defective in the PII mutants. To distinguish whether this process operates at the level of NR activity or whether the amount of nitrate reduction is controlled by nitrate uptake, the dependence of nitrate utilization on active transport was bypassed by increasing the nitrate concentration in the medium to 60 mM, a concentration at which nitrate enters the cells by diffusion (38). Under these conditions, nitrite formation was tested again at 10, 40, and 120 ␮mol photons s⫺1 m⫺2. If nitrate reduction is controlled by regulation of NR activity, no nitrite accumulation should occur. When, however, nitrate transport controls nitrate reduction, the wild-type cells should lose control. As shown in Fig. 7, wild-type cells were indeed no longer able to prevent nitrite formation with decreasing PPFD, implying that light control of nitrate utilization operates at the level of nitrate uptake.

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FIG. 7. Nitrite formation by wild-type cells of Synechocystis sp. strain PCC 6803 incubated at an illumination of 10 (circles), 40 (squares), or 120 (triangles) ␮mol photons s⫺1 m⫺2 in BG11 medium containing 60 mM nitrate.

FIG. 6. (A) Consumption of 100 ␮M nitrite by cells of wild-type Synechocystis sp. strain PCC 6803 (squares) and the PphA-deficient mutant MPphA (diamonds). (B) Consumption of a mixture of 100 ␮M nitrate (filled symbols) and 100 ␮M nitrite (open symbols) by wild-type Synechocystis sp. strain PCC 6803 cells (squares) and the PphA-deficient mutant MPphA (diamonds). (C) Consumption of a mixture of 100 ␮M nitrate (filled symbols) and 100 ␮M nitrite (open symbols) by NR-poisoned cells of wild-type Synechocystis sp. strain PCC 6803 (squares) and of the PphA-deficient mutant MPphA (diamonds). The assays were performed at an illumination of 10 ␮mol photons s⫺1 m⫺2. The data are from a representative of three independent experiments yielding nearly identical results.

DISCUSSION The present study reveals a novel regulatory link between photosynthetic electron transport and nitrate utilization in the cyanobacterium Synechocystis sp. strain PCC 6803. During photoautotrophic growth, assimilation of nitrate is a genuine photosynthetic process through the dependence of nitrate and nitrite reductases on PSI-reduced ferredoxin (4). The majority of photosynthetically generated reductant is required for CO2 fixation. In nitrate-grown cells, approximately 20% of reductant is utilized for conversion of nitrate to ammonium. At low PPFD, the availability of reductant limits assimilatory reac-

tions, and nitrate and nitrite reductases compete for limiting reductant. To avoid the accumulation of the intermediary product nitrite, nitrite reduction has to keep pace with nitrate reduction. In Synechocystis sp. strain PCC 6803 wild-type cells, no nitrite excretion occurs when the ambient nitrate concentration is so low that nitrate uptake depends on active transport. However, when the environmental concentration of nitrate rises to high levels, nitrite excretion occurs preferentially at lower light intensities. Under these conditions, nitrate enters the cells in a nitrate transport-independent manner (38). This implies that under low-light conditions, the in vivo activity of nitrate reductase exceeds nitrite reductase activity, resulting in excess formation of nitrite, which is then excreted to the medium. In contrast, no nitrite formation occurs in the presence of submillimolar concentrations of nitrate. From this it follows that Synechocystis wild-type cells regulate the reduction of nitrate in response to low light at the level of nitrate uptake. This is the same regulatory principle the cells use to control nitrate utilization in response to ammonium (27). Nitrate transport is rapidly inhibited upon ammonium treatment, a process that depends on the PII signal transduction protein, whereas nitrate reductase activity is not affected (27). However, the dependence of nitrate transport on the PII protein differs with respect to mode of signal transduction. Whereas the present study revealed that nonphosphorylated PII is necessary for the lowlight control of nitrate uptake (see below), regulation of nitrate uptake in response to ammonium does not depend on the phosphorylation state of PII. This was previously shown in mutants of Synechococcus elongatus PCC 7942 in which the site of phosphorylation, Ser49, was mutated to aspartate or glutamate (28) and was recently corroborated in Synechocystis sp. strain PCC 6803 (27). In accord with these data, this study shows that the PphA-deficient mutant, which is impaired in rapid PII-P dephosphorylation, is not affected in ammoniumresponsive control of nitrate uptake, whereas the PII-deficient mutant has lost this control. Most likely, binding or dissociation of effector molecules to PII is sufficient to mediate this response. In contrast to ammonium response, the PII-deficient and PphA-deficient mutants exhibit the same phenotype with respect to nitrate utilization under low-light conditions. In these mutants, conditions of limiting PPFD lead to the excretion of


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nitrite, even at low nitrate concentrations. Nitrite formation is the typical phenotype of an impaired nitrite reductase activity (48). Indeed, it could be shown that in spite of almost identical activity levels of NR and NiR, in vivo nitrite utilization is impaired under low-light conditions when nitrate is present. This impairment results from the competing activity of nitrate reductase under conditions of limiting reductant: when nitrate reductase was poisoned with tungstate, in vivo nitrite reduction in the presence of nitrate was not affected, and nitrite excretion could also be lowered by increasing the availability of reductant. In the PphA-deficient mutant, the PII signaling protein is present at wild-type levels. Moreover, PII signaling processes, which do not depend on the phosphorylation state of PII, are not affected in this mutant (26). For this reason, the mutant allows the identification of cellular processes in which the phosphorylation state of PII plays a role. The fact that nitrite excretion at low PPFD occurs in MPphA as well as in PIInull mutants implies that nonphosphorylated PII is required for this regulatory process. Indeed, the nonphosphorylated form of PII could be detected in wild-type cells under those conditions, where the mutants excreted nitrite. The inability to control nitrate utilization effectively explains the observed competitive disadvantage to wild-type cells. By excreting nitrite, the mutant wastes reductant, which can be used by wild-type cells. In mechanistic terms, the different regulation of nitrate transport by PII, which is independent of its phosphorylation state in response to ammonium but is dependent on nonphosphorylated PII in response to low light, remains elusive. However, it reminds one of the different regulation of NRT activities toward either ammonium or CO2 fixation. In a C-terminal truncated NrtC mutant of Synechococcus sp. strain PCC 7942 (43), the response of NRT activity toward ammonium was lost, whereas regulation in response to CO2 fixation was still functional, indicating the existence of at least two independent mechanisms in the regulation of nitrate transport. Biochemical analyses with purified components are necessary to resolve this issue and to reveal a potential link to PII signaling. As shown in this study, low PPFD results in a preferential dephosphorylation of PII by PphA. Decreasing 2-oxoglutarate or ATP levels are likely to be the primary signals to which PphA-mediated PII dephosphorylation responds (6, 44). Although subtle changes in the effector molecule levels may not be sufficient to allow a direct regulation of PII targets independent of the PII phosphorylation status, they may be sufficient to modulate the reactivity of PII-P toward PphA. In this context, it is interesting that the amount of PphA increases in the presence of nitrite (26), which might contribute to efficient dephosphorylation of PII under conditions of nitrite accumulation. Nonphosphorylated PII, in turn, controls nitrate transport to prevent further production of nitrite. Therefore, one function of PphA would be to amplify subtle changes in effector molecule concentrations by adjusting the phosphorylation status of PII, leading to a fine-tuned regulation of nitrate utilization. ACKNOWLEDGMENTS We are indebted to S. Bedu (Marseille) for providing the PII-deficient Synechocystis strain ⌬PII. Excellent technical assistance by Carmen Haas is gratefully acknowledged. We thank Gary Sawers for critically reading the manuscript. This work was supported by a grant from the DFG (Fo 195/4).

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