Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kloft, N. Articles by Forchhammer, K. Search for Related Content PubMed PubMed Citation Articles by Kloft, N. Articles by Forchhammer, K.

 Previous Article  |  Next Article 

Journal of Bacteriology, October 2005, p. 6683-6690, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6683-6690.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Signal Transduction Protein P_II Phosphatase PphA Is Required for Light-Dependent Control of Nitrate Utilization in Synechocystis sp. Strain PCC 6803

Nicole Kloft and Karl Forchhammer^*

Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany

Received 18 May 2005/ Accepted 18 July 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Signal transduction protein P_II is dephosphorylated in Synechocystis sp. strain PCC 6803 by protein phosphatase PphA. To determine the impact of PphA-mediated P_II dephosphorylation on physiology, the phenotype of a PphA-deficient mutant was analyzed. Mutants lacking either PphA or P_II 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 P_II 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 P_II 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 NtcA-activated gene expression (5). A central molecule for perception and signaling of the cellular nitrogen status in bacteria is the P_II signal transduction protein (2, 7, 37). The P_II protein family is one of the most widely distributed families of signal transduction proteins, whose members are present in all domains of life (2). P_II 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 P_II proteins is highly conserved, and in all cases analyzed so far, the P_II proteins recognize adenylate nucleotides and 2-oxoglutarate (9, 24, 30, 46), which control the reactivity of P_II towards various targets (23, 31). Furthermore, P_II 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 P_II proteins from proteobacteria), which further modulates signal output by P_II. 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).

P_II 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, P_II is almost completely nonphosphorylated. In nitrate-supplemented cells, intermediate levels of P_II phosphorylation, which are modulated by the inorganic carbon supply to the cells, are observed, and the highest levels of P_II phosphorylation occur under conditions of nitrogen starvation (7, 12). Targets of P_II signaling are beginning to emerge. Previously, it was shown in a P_II-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 component of NRT was regulated by P_II. Studies with S49 mutants of P_II, which potentially mimic the phosphorylated protein, showed that NRT was regulated by P_II even without changing the P_II modification status (28). A recent study confirmed that the phosphorylation status of P_II 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, high-affinity bicarbonate uptake is also regulated by P_II without requiring P_II modification. Moreover, NtcA-activated gene expression under conditions of nitrogen starvation was shown to depend on P_II signaling (1, 41). In these cases, the direct targets of interaction with P_II are not yet known at the molecular level. Recently, N-acetyl-L-glutamate kinase (NAGK) was identified as the first molecular target of P_II signaling in a cyanobacterium (3, 15). NAGK catalyzes the first committed step in arginine biosynthesis and forms a tight complex with nonphosphorylated P_II. Binding of P_II strongly enhances the catalytic activity of this enzyme, whereas no NAGK activation or complex formation occurs with S49-modified P_II (15, 34).

The cellular signal for P_II 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 P_II protein (P_II-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 P_II-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 P_II-P dephosphorylation. A PphA-deficient mutant was unable to rapidly dephosphorylate P_II-P in response to various signals (7, 22), in agreement with the finding that P_II-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 P_II phosphorylation/dephosphorylation cycle in this cyanobacterium. Here, we describe a new regulatory mechanism in which P_II-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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and growth conditions. Synechocystis sp. strain 6803^T (13), the derived PphA-deficient mutant MPphA (pphA::kan) (22), and the P_II null mutant P_II (glnB::spec) (19) were grown in liquid BG11 medium (42) supplemented with 5 mM NaHCO₃ and 17.6 mM NaNO₃ (BG11^N) 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 µmol photons s^–1 m^–2 from white fluorescent tubes (LUMILUX de Luxe Daylight, Osram). The mutant MPphA was maintained with kanamycin (30 µg ml^–1) and the P_II mutant with spectinomycin (35 µg 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 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 µmol 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 BG11^N 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 µmol 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-µg 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 BG11^N 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 µM 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 P_II were grown in modified BG11 medium containing nitrite (5 mM final concentration), in which molybdate was replaced by tungstate (4.8 µM). 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 µM) was added.

Determination of the modification state of P_II. The phosphorylation state of P_II in vivo was analyzed by nondenaturing polyacrylamide gel electrophoresis followed by immunoblot analysis of P_II as described previously (10). P_II⁰, P_II¹, P_II², and P_II³ represent isoforms of the trimeric P_II 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Growth phenotype of a PphA-deficient mutant. Previous investigations showed that PphA is required for the rapid dephosphorylation of P_II-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 P_II, 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 question of the physiological significance of PphA-mediated dephosphorylation of P_II-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 nitrate-limited mixed culture revealed the gradual disappearance of the PphA mutant while the relative proportion of wild-type cells increased (Fig. 1B and C).

View larger version (29K): [in this window] [in a new window]

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.

Nitrate utilization and nitrite excretion in mutants of the P_II 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 P_II-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).

View larger version (13K): [in this window] [in a new window]

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 mid-exponential 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.

To analyze the rate of nitrate consumption and concomitant formation of nitrite in vivo, cells were incubated in medium containing 200 µM 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 P_II-deficient strain (P_II), 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 P_II protein is required for the control of nitrate utilization in response to a short-term exposure to ammonium (19, 27, 29), albeit by a mechanism that does not require P_II dephosphorylation.

View larger version (18K): [in this window] [in a new window]

FIG. 3. (A) Consumption of 200 µM 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 µM 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 µmol photons s–1 m–2. A representative of three independent experiments is shown.

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, 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 wild-type 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 µg for the wild type and 5.0 µg 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 P_II were incubated in medium containing 200 µM NaNO₃ at PPFDs of 10, 40, and 120 µmol 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 P_II 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 µmol photons s^–1 m^–2, MPphA converted only 13% and P_II 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 P_II 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 µE), an intermediate level of P_II phosphorylation was observed, with appreciable levels of the nonphosphorylated form (P_II⁰) and predominantly the forms with one and two phosphorylated subunits (P_II¹ and P_II²). At higher light intensity, the phosphorylation status was clearly increased, with only traces of unmodified P_II and high levels of fully phosphorylated P_II (P_II³). By contrast, in MPphA cells, P_II protein was present in its highly phosphorylated forms (P_II² and P_II³) with no unmodified isoform detectable under any light conditions.

View this table: [in this window] [in a new window]

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

View larger version (42K): [in this window] [in a new window]

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 µmol photons s–1 m–2), samples were collected and the phosphorylation state of PII was analyzed.

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 P_II. 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 µM NaNO₃, and the other half was incubated in medium containing 200 µM NaNO₃ together with 0.1% glucose at an illumination of 10 µmol 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 P_II under those conditions is shown in Fig. 5. In the absence of glucose, three phosphorylated P_II isoforms were observed in wild-type cells, the unmodified form (P_II⁰) and the phosphorylated forms of P_II with one and two phosphorylated subunits (P_II¹ and P_II²), whereas only highly phosphorylated P_II forms (P_II² and P_II³) were observed in MPphA. The addition of glucose resulted in an increased phosphorylation of P_II, with P_II⁰ disappearing in cells of the wild type.

View this table: [in this window] [in a new window]

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

View larger version (25K): [in this window] [in a new window]

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 µmol photons s^–1 m^–2 in P_II 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 µM (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 P_II 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 P_II 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 P_II 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 µmol 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.

View larger version (15K): [in this window] [in a new window]

FIG. 6. (A) Consumption of 100 µM 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 µM nitrate (filled symbols) and 100 µM 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 µM nitrate (filled symbols) and 100 µM 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 µmol photons s–1 m–2. The data are from a representative of three independent experiments yielding nearly identical results.

View larger version (13K): [in this window] [in a new window]

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) µmol photons s–1 m–2 in BG11 medium containing 60 mM nitrate.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 reactions, 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 P_II signal transduction protein, whereas nitrate reductase activity is not affected (27). However, the dependence of nitrate transport on the P_II protein differs with respect to mode of signal transduction. Whereas the present study revealed that nonphosphorylated P_II is necessary for the low-light control of nitrate uptake (see below), regulation of nitrate uptake in response to ammonium does not depend on the phosphorylation state of P_II. 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 P_II-P dephosphorylation, is not affected in ammonium-responsive control of nitrate uptake, whereas the P_II-deficient mutant has lost this control. Most likely, binding or dissociation of effector molecules to P_II is sufficient to mediate this response.

In contrast to ammonium response, the P_II-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 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 P_II signaling protein is present at wild-type levels. Moreover, P_II signaling processes, which do not depend on the phosphorylation state of P_II, are not affected in this mutant (26). For this reason, the mutant allows the identification of cellular processes in which the phosphorylation state of P_II plays a role. The fact that nitrite excretion at low PPFD occurs in MPphA as well as in P_IInull mutants implies that nonphosphorylated P_II is required for this regulatory process. Indeed, the nonphosphorylated form of P_II 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 P_II, which is independent of its phosphorylation state in response to ammonium but is dependent on nonphosphorylated P_II 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 P_II signaling. As shown in this study, low PPFD results in a preferential dephosphorylation of P_II by PphA. Decreasing 2-oxoglutarate or ATP levels are likely to be the primary signals to which PphA-mediated P_II dephosphorylation responds (6, 44). Although subtle changes in the effector molecule levels may not be sufficient to allow a direct regulation of P_II targets independent of the P_II phosphorylation status, they may be sufficient to modulate the reactivity of P_II-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 P_II under conditions of nitrite accumulation. Nonphosphorylated P_II, 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 P_II, leading to a fine-tuned regulation of nitrate utilization.

 
We are indebted to S. Bedu (Marseille) for providing the P_II-deficient Synechocystis strain P_II. 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).

 
* Corresponding author. Mailing address: Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany. Phone: 49 641-9935545. Fax: 49 641-9935549. E-mail: Karl.Forchhammer{at}mikro.bio.uni-giessen.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Aldehni, M. F., J. Sauer, C. Spielhaupter, R. Schmid, and K. Forchhammer. 2003. Signal transduction protein P_II is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC 7942. J. Bacteriol. 185:2582-2591.[Abstract/Free Full Text]
2
2. Arcondeguy, T., R. Jack, and M. Merrick. 2001. P_II signal transduction proteins: pivotal players in microbial nitrogen control. Microbiol. Mol. Biol. Rev. 65:80-105.[Abstract/Free Full Text]
3
3. Burillo, S., I. Luque, I. Fuentes, and A. Contreras. 2004. Interactions between the nitrogen signal transduction protein P_II and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis. J. Bacteriol. 186:3346-3354.[Abstract/Free Full Text]
4
4. Flores, E., and A. Herrero. 1994. Assimilatory nitrogen metabolism and its regulation, p. 488-517. In D. A. Bryant (ed.), The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
5
5. Flores, E., A. M. Muro-Pastor, and A. Herrero. 1999. Cyanobacterial nitrogen assimilation genes and NtcA-dependent control of gene expression, p. 463-477. In G. Pescheck, W. Löffelhardt, and G. Schmetterer (ed.), The phototrophic prokaryotes. Kluwer Academic/Plenum Publishers, New York, N.Y.
6
6. Forchhammer, K., A. Irmler, N. Kloft, and U. Ruppert. 2004. P_II signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge. Physiol. Plant. 120:51-54.[CrossRef][Medline]
7
7. Forchhammer, K. 2004. Global carbon/nitrogen control by P_II signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol. Rev. 28:319-333.[CrossRef][Medline]
8
8. Forchhammer, K. 1999. The P_II protein in Synechococcus PCC 7942 senses and signals 2-oxoglutarate under ATP-replete conditions, p. 549-553. In G. Peschek, W. Löffelhardt, and G. Schmetterer (ed.), The phototrophic prokaryotes. Kluwer Academic/Plenum Publishers, New York, N.Y.
9
9. Forchhammer, K., and A. Hedler. 1997. Phosphoprotein P_II from cyanobacteria. Eur. J. Biochem. 244:869-875.[Medline]
10
10. Forchhammer, K., and N. Tandeau de Marsac. 1994. The P_II protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status. J. Bacteriol. 176:84-91.[Abstract/Free Full Text]
11
11. Forchhammer, K., and N. Tandeau de Marsac. 1995. Phosphorylation of the P_II protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity. J. Bacteriol. 177:5812-5817.[Abstract/Free Full Text]
12
12. Forchhammer, K., and N. Tandeau de Marsac. 1995. Functional analysis of the phosphoprotein P_II from the cyanobacterium Synechococcus sp. strain PCC 7942. J. Bacteriol. 177:2033-2040.[Abstract/Free Full Text]
13
13. Grigorieva, G., and S. V. Shestakov. 1982. Transformation in the cyanobacterium Synechocystis sp. PCC 6803. FEMS Microbiol. Lett. 13:367-370.[CrossRef]
14
14. Guerrero, M. G., and C. Lara. 1987. Assimilation of inorganic nitrogen, p. 163-186. In P. Fay and C. V. Baalen (ed.), The cyanobacteria. Elsevier Science Publishers, Amsterdam, The Netherlands.
15
15. Heinrich, A., M. Maheswaran, U. Ruppert, and K. Forchhammer. 2004. The Synechococcus elongatus P_II signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase. Mol. Microbiol. 52:1303-1314.[CrossRef][Medline]
16
16. Herrero, A., E. Flores, and M. G. Guerrero. 1981. Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719. J. Bacteriol. 145:175-180.[Abstract/Free Full Text]
17
17. Herrero, A., E. Flores, and M. G. Guerrero. 1985. Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp. FEMS Microbiol. Lett. 26:21-25.[CrossRef]
18
18. Herrero, A., and M. G. Guerrero. 1986. Regulation of nitrite reductase in the cyanobacterium Anacystis nidulans. J. Gen. Microbiol. 132:2463-2468.
19
19. Hisbergues, M., R. Jeanjean, F. Joset, N. Tandeau de Marsac, and S. Bedu. 1999. Protein P_II regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in Synechocystis PCC 6803. FEBS Lett. 463:216-220.[CrossRef][Medline]
20
20. Irmler, A. 2001. Thesis. Universität Giessen, Giessen, Germany.
21
21. Irmler, A., S. Sanner, H. Dierks, and K. Forchhammer. 1997. Dephosphorylation of the phosphoprotein P_II in Synechococcus PCC 7942: identification of an ATP and 2-oxoglutarate-regulated phosphatase activity. Mol. Microbiol. 26:81-90.[CrossRef][Medline]
22
22. Irmler, A., and K. Forchhammer. 2001. A PP2C-type phosphatase dephosphorylates the P_II signaling protein in the cyanobacterium Synechocystis PCC 6803. Proc. Natl. Acad. Sci. USA 98:12978-12983.[Abstract/Free Full Text]
23
23. Jiang, P., and A. J. Ninfa. 1999. Regulation of the autophosphorylation of Escherichia coli nitrogen regulator II by the PII signal transduction protein. J. Bacteriol. 181:1906-1911.[Abstract/Free Full Text]
24
24. Kamberov, E. S., M. A. Atkinson, and A. J. Ninfa. 1995. The Escherichia coli signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J. Biol. Chem. 270:17797-17807.[Abstract/Free Full Text]
25
25. Kaneko, T., S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N. Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi, A. Matsuno, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, C. Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, and S. Tabata. 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 30:109-136.
26
26. Kloft, N., G. Rasch, and K. Forchhammer. 2005. Protein phosphatase PphA from Synechocystis PCC 6803: the physiological framework of P_II-P dephosphorylation. Microbiology 151:1275-1283.[Abstract/Free Full Text]
27
27. Kobayashi, M., N. Takatani, M. Taniwaga, and T. Omata. 2005. Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 187:498-506.[Abstract/Free Full Text]
28
28. Lee, H.-M., E. Flores, K. Forchhammer, A. Herrero, and N. Tandeau de Marsac. 2000. Phosphorylation of the signal transducer P_II protein and an additional effector are required for the P_II-mediated regulation of nitrate and nitrite uptake in the cyanobacterium Synechococcus sp. PCC 7942. Eur. J. Biochem. 267:591-600.[Medline]
29
29. Lee, H.-M., E. Flores, A. Herrero, J. Houmard, and N. Tandeau de Marsac. 1998. A role for the signal transduction protein P_II in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Lett. 427:291-295.[CrossRef][Medline]
30
30. Little, R., F. Reyes-Ramirez, Y. Zhang, W. C. van Heeswijk, and R. Dixon. 2000. Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the P_II regulatory protein. EMBO J. 19:6041-6050.[CrossRef][Medline]
31
31. Little, R., V. Colombo, A. Leech, and R. Dixon. 2002. Direct interaction of the NifL regulatory protein with the GlnK signal transducer enables the Azotobacter vinelandii NifL-NifA regulatory system to respond to conditions replete for nitrogen. J. Biol. Chem. 277:15472-15481.[Abstract/Free Full Text]
32
32. Luque, I., A. Herrero, E. Flores, and F. Madueno. 1992. Clustering of genes involved in nitrate assimilation in the cyanobacterium Synechococcus. Mol. Gen. Genet. 232:7-11.[CrossRef][Medline]
33
33. Maeda, S., and T. Omata. 1997. Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite. J. Biol. Chem. 272:3036-3041.[Abstract/Free Full Text]
34
34. Maheswaran, M., C. Urbanke, and K. Forchhammer. 2004. Complex formation and catalytic activation by the P_II signaling protein of N-acetyl-L-glutamate kinase from Synechococcus elongatus strain PCC 7942. J. Biol. Chem. 53:55202-55210.
35
35. Manzano, C., P. Candau, C. Gomez-Moreno, A. M. Relimpio, and M. Losada. 1976. Ferredoxin-dependent photosynthetic reduction of nitrate and nitrite by particles of Anacystis nidulans. Mol. Cell. Biochem. 10:161-169.[CrossRef][Medline]
36
36. Muro-Pastor, I., J. C. Reyes, and F. J. Florencio. 2001. Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J. Biol. Chem. 276:38320-38328.[Abstract/Free Full Text]
37
37. Ninfa, A. J., and M. R. Atkinson. 2000. P_II signal transduction proteins. Trends Microbiol. 8:172-179.[CrossRef][Medline]
38
38. Omata, T., M. Ohmori, N. Aria, and T. Ogawa. 1989. Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport. Proc. Natl. Acad. Sci. USA 86:6612-6616.[Abstract/Free Full Text]
39
39. Omata, T. 1991. Cloning and characterization of the nrtA gene that encodes a 45-kDa protein involved in nitrate transport in the cyanobacterium Synechococcus PCC 7942. Plant Cell Physiol. 32:151-157.[Abstract/Free Full Text]
40
40. Omata, T., X. Andriesse, and A. Hirano. 1993. Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus PCC 7942. Mol. Gen. Genet. 236:193-202.[CrossRef][Medline]
41
41. Paz-Yepez, J., E. Flores, and A. Herrero. 2003. Transcriptional effects of the signal transduction protein P_II (glnB product) on NtcA-dependent genes in Synechococcus sp. PCC 7942. FEBS Lett. 543:42-46.[CrossRef][Medline]
42
42. Rippka, R. 1988. Isolation and purification of cyanobacteria. Methods Enzymol. 167:3-27.[Medline]
43
43. Rodriguez, R., M. Kobayashi, T. Omata, and C. Lara. 1998. Independence of carbon and nitrogen control in the posttranscriptional regulation of nitrate transport in the cyanobacterium Synechococcus sp. strain PCC 7942. FEBS Lett. 432:207-212.[CrossRef][Medline]
44
44. Ruppert, U., A. Irmler, N. Kloft, and K. Forchhammer. 2002. The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein P_II. Mol. Microbiol. 44:855-864.[CrossRef][Medline]
45
45. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
46
46. Smith, C., A. M. Weljie, and G. B. G. Moorhead. 2003. Molecular properties of the putative nitrogen sensor P_II from Arabidopsis thaliana. Plant J. 33:353-360.[CrossRef][Medline]
47
47. Snell, F. D., and C. T. Snell. 1949. Colorimetric methods of analysis, p. 804-805. Van Nostrand, New York, N.Y.
48
48. Suzuki, I., N. Horie, T. Sugiyama, and T. Omata. 1995. Identification and characterization of two nitrogen-regulated genes of the cyanobacterium Synechococcus sp. strain PCC7942 required for maximum efficiency of nitrogen assimilation. J. Bacteriol. 177:290-296.[Abstract/Free Full Text]

---

Journal of Bacteriology, October 2005, p. 6683-6690, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6683-6690.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Espinosa, J., Castells, M. A., Laichoubi, K. B., Contreras, A. (2009). Mutations at pipX Suppress Lethality of PII-Deficient Mutants of Synechococcus elongatus PCC 7942. J. Bacteriol. 191: 4863-4869 [Abstract] [Full Text]  
• Osanai, T., Tanaka, K. (2007). Keeping in Touch with PII: PII-Interacting Proteins in Unicellular Cyanobacteria. Plant Cell Physiol 48: 908-914 [Abstract] [Full Text]  
• Espinosa, J., Forchhammer, K., Contreras, A. (2007). Role of the Synechococcus PCC 7942 nitrogen regulator protein PipX in NtcA-controlled processes. Microbiology 153: 711-718 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kloft, N. Articles by Forchhammer, K. Search for Related Content PubMed PubMed Citation Articles by Kloft, N. Articles by Forchhammer, K.