Effect of Nitrogen Source on Cyanophycin Synthesis in Synechocystis sp. Strain PCC 6308
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《细菌学杂志》
Departments of Chemistry,Biological Sciences, Wellesley College, Wellesley, Massachusetts 02481
ABSTRACT
Experiments were carried out to examine the effects of nitrogen source on nitrogen incorporation into cyanophycin during nitrogen limitation and repletion, both with or without inhibition of protein synthesis, in cyanobacteria grown on either nitrate or ammonium. The use of nitrate and ammonium, 14N labeled in the growth medium and 15N labeled in the repletion medium, allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance spectroscopy. The data suggest that nitrogen from both the breakdown of cellular protein (14N) and directly from the medium (15N) is incorporated into cyanophycin. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells and to the possible expression of cyanophycin synthetase during nitrogen starvation.
INTRODUCTION
Cyanobacteria are capable of growing on a number of different nitrogen sources. Nitrate, nitrite, ammonium, urea, several amino acids, atmospheric nitrogen, nucleosides, and bases all have been reported to be assimilated by some cyanobacteria, but ammonium appears to be assimilated preferentially (7, 17). Nitrate is probably the most abundant form of combined nitrogen available for assimilation by cyanobacteria (10). The nondiazotrophic cyanobacterium Synechocystis sp. strain PCC 6308 has been shown to metabolize nitrate, ammonium, and diamino acids (4) and to incorporate the nitrogen from these sources into necessary metabolites, as well as into the nitrogen storage material, cyanophycin granule polypeptide (cyanophycin). Nitrogen limitation in nondiazotrophic cyanobacteria leads to many different effects that have been described in a variety of strains (4, 30).
Utilization of different nitrogen sources first requires their passage through the permeability barrier of the cytoplasmic membrane into the cyanobacterial cell. At low nitrate concentrations, the endergonic uptake of nitrate in freshwater cyanobacteria takes place through ABC-type transporters that exhibit high affinity for nitrate (27). Passive diffusion of nitrate takes place in Synecchococcus sp. strain PCC 7942 at nitrate concentrations above 1 mM (26). Ammonium at low concentrations (<0.25 mM) is taken up by a permease that catalyzes a membrane potential-dependent active transport, but this system is repressed in higher concentrations of ammonium where passive diffusion of unprotonated ammonia may be the mechanism for net uptake of ammonium (24). After diffusion, intracellular ammonium is trapped by glutamine synthetase. All nitrogen sources that are assimilated are converted to intracellular ammonium. Intracellular nitrate, for example, is reduced to nitrite by nitrate reductase; the nitrite is then reduced to ammonium by nitrite reductase. The ammonium then enters the glutamine synthetase/glutamate synthase cycle to be incorporated into 2-oxoglutarate (21, 24, 25, 35).
Many forms of transcriptional and posttranslational control have been shown to be involved in nitrogen assimilation when cells are grown under different physiological conditions (reviewed in references 11 and 12). A global nitrogen-regulating gene, ntcA, has been shown to synthesize a transcriptional regulator whose activity is influenced by 2-oxoglutarate. This gene is activated by the ATP binding, signal transduction protein PII (glnB gene product [13]) especially when the C-to-N ratio in the cell is high (21, 28), as is true in cases of nitrogen limitation. PII in its phosphorylated form also is required for the activation of transcription of NtcA-dependent genes, such as nitrate reductase, under conditions of nitrogen limitation (1). However, when cells are placed in conditions where there are low 2-oxoglutarate levels (high N-to-C levels), the nonphosphorylated form of PII appears to cause inhibition of the nitrate/nitrite permease (18). In nitrogen-starved Synechocystis sp. strain PCC 6803, the expression of three ammonium permeases was higher than when cells were incubated in either ammonium or nitrate (24). Similarly, glutamine synthetase in Synechocystis sp. strain PCC 6803 is inactivated in the presence of ammonium by two small protein factors (IF7 and IF17); these factors are regulated by NtcA (14).
Two molecules, both with high nitrogen content, appear to be nitrogen storage compounds in cyanobacteria: phycocyanin and cyanophycin. Phycocyanin is the principal accessory pigment in most cyanobacteria, whereas cyanophycin is thought to function primarily as a nitrogen reserve. Cyanophycin, a non-ribosomally synthesized peptide, composed of arginine and aspartic acid, accumulates in cyanobacteria when they are grown under all unbalanced nutrient conditions except nitrogen starvation (33, 3). Cyanobacteria that synthesize cyanophycin tend to use it as a nitrogen source during nitrogen starvation before using phycocyanin and other sources of cellular nitrogen such as proteins (5).
There is evidence that cyanophycin acts as transient store for newly fixed nitrogen in the heterocysts of diazotrophic cyanobacteria (19), and several studies have suggested that cyanophycin is a dynamic reservoir in most cyanobacteria (8, 22, 20). When cyanobacteria are starved for nitrogen, cyanophycin is broken down; when nitrogen becomes available, cyanophycin is again synthesized. Upon reintroduction of nitrogen, cells rebuild their nitrogen stores and begin to grow. It appears that cyanophycin may play an even more important role as a nitrogen reserve in diazotrophic unicellular strains (20) than in nondiazotrophic unicellular strains such as Synechocystis sp. strain PCC 6803, where phycobilisomes appear to be the main nitrogen reserve. Picossi et al. (29) showed that expression of the genes for cyanophycin metabolism (cphA for cyanophycin synthesis and cphB for cyanophycinase) increases in the absence of ammonium in Anabaena sp. strain PCC 7120. The inability to degrade cyanophycin is detrimental to the diazotrophic growth of this strain.
Cyanophycin is synthesized in Synechocytis sp. strain PCC 6308 immediately after nitrogen is replenished (4), whereas phycocyanin is not synthesized until about 6 h later. The synthesis of cyanophycin peaks at up to 12% of the dry weight of the cells by 10 to 12 h and then decreases to the low levels found in typical exponential growth. After growth of the same strain in medium containing ammonium, and then starvation and replenishing with ammonium, Mackerras et al. (22) also found that cyanophycin was synthesized immediately and then degraded within 24 h. A salt-sensitive, glycoprotease-negative mutant (gcp mutant) of Synechocystis sp. strain PCC 6803 accumulated cyanophycin to a level 40 times that of the wild-type strain since it was unable to remobilize cyanophycin (37). In this mutant, phycobilisomes were degraded as a source of nitrogen.
Recently, we have shown that cells grown in low light with or without the protein synthesis inhibitor chloramphenicol (CM), as well as cells grown in normal light with CM, synthesize cyanophycin from nitrogen obtained both from degradation of cellular proteins and directly from the medium (6). Nitrogen from the medium was incorporated at a faster rate and to a greater extent than nitrogen from protein degradation.
Since cyanophycin is a nitrogen storage molecule that may be a dynamic reserve, the experiments described here were designed to determine how the source of nitrogen affects whether cyanophycin is synthesized from nitrogen derived from proteolysis of cellular protein or from nitrogen in the cells' growth medium. Experiments were carried out here to examine the effects of nitrogen limitation on nitrogen incorporation into cyanophycin with or without inhibition of protein synthesis in cells growing on either nitrate or ammonium. Cells were grown in 14N medium and then either washed or allowed to deplete their nitrogen before being transferred into 15N repletion medium. The use of 14N in the growth medium and 15N in the repletion medium allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance (NMR) spectroscopy (34). In all cases, the nitrogen in cyanophycin came from both the breakdown of cellular protein and nitrogen in the medium but at different rates and to different extents depending on the nitrogen source and stress condition.
MATERIALS AND METHODS
BG-11 medium (2) containing either 17.6 mM sodium nitrate (Na14NO3) or 10 mM ammonium chloride (14NH4Cl) and 2 mM TES (pH 10) was used for the growth and maintenance of cells. Experimental cells were grown at 35°C with cool white fluorescent light of approximately 80 μE s–1 m–2 in BG-11 medium containing 22.6 mM sodium carbonate in 1-liter Roux flasks that were continuously bubbled with 5% CO2 in air. Cells were starved for nitrogen by transferring exponentially growing cells to BG-11 medium containing the 14N forms of either 0.44 mM sodium nitrate or 1 mM ammonium chloride. Nitrogen starvation was observed by the culture's change in color from green to yellow, a process that usually required about 3 days. When starved, the culture was divided and added to new Roux flasks where cells were replenished with 10 mM 15NH4Cl (Cambridge Isotopes, Cambridge, MA) and 2 mM TES or with 5 to 17.6 mM Na15NO3 (Cambridge Isotopes). In some experiments, 5 μg of CM/ml was added at the same time as the nitrogen source. In experiments in which cultures were not nitrogen starved, cells were washed before the 15N source was added.
Cyanophycin was extracted from cells harvested immediately and at increasing times up to 24 h after transfer from starvation conditions or washing to 15N growth medium, either with or without CM, and dissolved in 0.1 M HCl as described by Allen et al. (6). 1H NMR spectra of all cyanophycin samples were run on a Bruker Avance spectrometer (Bruker Instruments, Billerica, MA) at 400.152 MHz (34, 6). Spectra were calibrated to a 0.010 M solution of TSP (3-trimethylsilyl-2,2,3,3-tetradeutero sodium propionate; Cambridge Isotopes) in D2O (Aldrich Chemical Co., Milwaukee, WI) and analyzed as described in Allen et al. (6), using the 1D WIN-NMR PC program (Bruker Instruments, Billerica, MA). In some cases only the NH peaks at 6.8 ppm (, '-14N, or 15N) and 7.4 ppm (-14N or 15N) were integrated because the small chemical shift difference between the -NH peaks for Asp and Arg (8.4 to 8.5 ppm) made the multiplets difficult to resolve and integrate accurately. Ratios of integration of peaks due to protons bonded to each 14N and 15N were divided by the sum of the integrations of the peaks for protons bonded to both nitrogen isotopes to calculate the percentages of each isotopic form of cyanophycin present at each time point. Total cyanophycin was determined by integrating the arginine -CH proton peak at 3.25 ppm (9). These integrations were standardized to the TSP integration and then corrected for the volume of the sample. Amounts of cyanophycin containing 14N and 15N at each time point were thus the product of the total cyanophycin and the fraction of each isotopic species.
Non-nitrogen-starved cells have different amounts of cyanophycin present when they are washed and placed into new nitrogen-containing medium, depending on their growth stage. Exponentially growing cells have small, but sometimes measurable amounts of cyanophycin when transferred. The amount of cyanophycin present in these cells (determined from the arginine -CH peak at 3.25 ppm) at the time of transfer was subtracted from the amount of cyanophycin at each point in time course experiments in order to calculate rates and times of incorporation of 14N and 15N comparable to the starved cell experiments. Without this correction, rates of 15N incorporation would have appeared lower and the time at which 15N=14N would have appeared later. Nitrogen-starved cells had no cyanophycin at the time of medium switch.
The concentration of chlorophyll a in the cultures was measured by adding 1 ml of methanol to 50 μl of culture, microfuging the mixture for 3 min, and measuring the absorbance of the supernatant at 665 nm. Pigments were also observed by recording whole-cell visible spectra between 400 and 750 nm using an opal glass technique (32). Nitrate reductase assays were performed at various times on cells grown with either nitrate or ammonium, with or without the addition of CM, as described by Herrero et al. (16). Cells were permeabilized by using toluene and incubated for 5 min at 30°C with substrate, methyl viologen, and sodium dithionite. The reaction was stopped and the colored nitrite complex developed by addition of sulfanilamide in HCl and N-(1-naphthyl)-ethylenediamine.
RESULTS
The quantitative data used to calculate the incorporation of nitrogen into the cyanophycin of Synechocytis sp. strain PCC 6308 under different stress conditions were derived from 1H NMR spectra of isolated cyanophycin solubilized in 0.1 M HCl. Figure 1 shows a typical series over time of 1H NMR spectra of cyanophycin extracted from cells grown in medium containing 14NH4+, starved for nitrogen, and then replenished in medium containing 15NH4+ and CM. All cyanophycin peaks are labeled. Note that with increasing time the peaks to the left of the large central water peak (protons bonded to nitrogens) became more complex, indicating the incorporation of 15N into the cyanophycin. Also note that these peaks and the peaks to the right of the water peak (protons bonded to carbons) increased in intensity with time, indicating production of cyanophycin. The size of the TSP peak was used to normalize all spectra. The large central peak is that of H2O, which, although suppressed by the NMR pulse sequence used, is still much larger than the peaks of cyanophycin.
Plots of the percentages of 14N and 15N cyanophycin for the same experiment are shown in Fig. 2. The graph shows data from the integration of all of the NH peaks for both nitrogen isotopes. The time at which the percentages of 14N and 15N cyanophycin became equal was between 0.5 and 1 h, showing that 15N was incorporated into cyanophycin very rapidly and that by 24 h 90% of the cyanophycin was labeled with medium nitrogen (15N). Using the percentages of the two nitrogen isotopes and the amount of cyanophycin at each time point, the rates of production of 14N and 15N cyanophycin were calculated and are shown in Fig. 3. The slopes of these lines give the relative rates of production for the two isotopic molecules, representing the amount of nitrogen in cyanophycin from breakdown of cellular protein (14N) or from the medium (15N). In this ammonium/starve/ammonium with CM experiment there was little incorporation of nitrogen from the breakdown of cellular protein during the 24 h of the experiment, whereas the rate of cyanophycin synthesis from medium nitrogen increased rapidly before 2 h. The ratio of rates of incorporation of 15N to 14N was 11:1.
The results of 1H NMR spectroscopy studies of the cyanophycin produced over time under 12 combinations of nitrogen source, stress, and replenishing conditions are shown in Tables 1 and 2. Each set of data was obtained from graphs similar to those in Fig. 1, 2, and 3. Table 1 is organized to systematically compare the effects on nitrogen-starved cells of CM and nitrogen source during growth and repletion. Table 2 compares the effects of nitrogen source on nitrogen-replete cells treated with CM. In half the cases shown in Table 1 the nitrogen source for replenishment remained the same as before starvation, while in the other half the source was switched.
Table 1 shows that the highest rates of incorporation of medium nitrogen into cyanophycin were observed in the ammonium/starve/ammonium experiments and the ammonium/starve/ammonium plus CM experiments where cells immediately took up ammonium from the medium and incorporated it into cyanophycin after replenishment. The ratio of rates of 15N:14N incorporation into cyanophycin were the highest in any of the 12 experiments at 19:1 and 11:1. The results in Table 1 for nitrogen-starved cells grown on ammonium and replenished with nitrate in the absence of CM show that cyanophycin is synthesized more slowly, incorporation of medium nitrogen (15N) occurs to a lesser extent, and the relative rates of incorporation of medium and protein nitrogen are lower when the nitrogen source is switched to nitrate after starvation than when it remains ammonium. For starved nitrate-grown cells replenished in the absence of CM the effects of nutrient switch are much smaller. When nitrogen-starved cells grown on ammonium were replenished with nitrate in the presence of CM the extent of medium nitrogen incorporation and the ratio of rates of incorporation of medium and protein nitrogen increased. CM had little effect when the nitrogen source was switched from nitrate to ammonium or was kept constant for nitrogen-starved cells.
Table 2 shows the response of nitrogen-replete cells to CM when the nitrogen source was the same or was switched. Nitrate-grown, nitrogen-replete cells utilized external nitrate or ammonium at approximately equal rates when treated with CM, but ammonium-grown nitrogen-replete cells utilized external ammonium slowly and external nitrate even more slowly when CM was present. For all of these nitrogen-replete cells treated with CM there was a lag before any new cyanophycin was synthesized, except in the case of cells grown initially in nitrate and replenished with nitrate.
Nitrate reductase activity was found to be significantly higher, by at least an order of magnitude, in cells growing in nitrate than in those growing in ammonium (t = 5.79, P = 3.99E-06). Figure 4 shows the activity of nitrate reductase expressed as μg of nitrite produced/μg of chlorophyll. Nitrate reductase activity was low in ammonium-grown cells but increased as ammonium was depleted from the medium during starvation. When starved cells were replenished with ammonium, enzyme activity decreased to its prestarvation level. When starved cells were replenished with nitrate, nitrate reductase activity increased dramatically until between 22 and 46 h after replenishing, after which activity began to decrease. When CM was added with nitrate at the time of replenishing nitrate-starved cells, nitrate reductase activity remained low for at least 5 h (data not shown), suggesting that the increase in nitrate reductase activity requires new protein synthesis.
DISCUSSION
Carr (8) first suggested that nitrogen might move in and out of cyanophycin during cell growth. It was later shown that cyanophycin constitutes a dynamic reservoir of fixed nitrogen in diazotrophic cyanobacteria (31). Using cyanophycin synthetase mutants, it was recently demonstrated that the path of newly synthesized organic nitrogen through cyanophycin in the heterocyst is not obligatory (36, 29). The purpose of the present experiments was to follow the path of nitrogen into cyanophycin when a nondiazotrophic cyanobacterium was grown in the presence of each of two of its common nitrogen sources: nitrate or ammonium.
Using 1H NMR spectroscopy of cyanophycin from cells grown first in medium with a 14N nitrogen source, followed by transfer into 15N medium, allows the quantification of amounts of nitrogen in the macromolecule from both sources. In these experiments, Synechocystis sp. strain PCC 6308, which forms large amounts of cyanophycin (5), was grown in either 14N nitrate or in 14N ammonium to exponential phase. Cells were then either starved for nitrogen or not starved. If starved, cells would have little or no cyanophycin and cellular proteins would be degraded. Cells that were not starved would have small, but varying amounts of cyanophycin and more 14N cellular protein. In the presence of CM, new protein synthesis would be prevented. New cyanophycin synthesis would thus have to come largely from the medium nitrogen (15N) directly, or from previously synthesized protein (14N), without going through newly synthesized proteins in the cell.
The results from these experiments suggest that nitrogen for cyanophycin synthesis comes preferentially from the external medium unless this nitrogen cannot be used due to either none being present or to the absence of the enzymes necessary for its utilization. Furthermore, since cyanophycin appears to be synthesized more slowly when the nitrogen source is switched after starvation than when it remains the same (Table 1), either new enzyme synthesis or enzyme activation is necessary. Nitrate reductase activity was very low when ammonium was present in the medium (Fig. 4), a finding in agreement with data for other strains of cyanobacteria (16). Thus, when nitrate was added to ammonium-grown cells without prior nitrogen starvation, low levels of nitrate reductase would be present and little nitrate could reach cyanophycin.
Lack of nitrate reductase activity in nitrogen-replete cells grown in ammonium medium before being transferred to nitrate and CM was hypothesized as the factor responsible for the very late and limited incorporation of medium nitrogen into cyanophycin (Table 2). Figure 4 shows that nitrate reductase activity was very low when ammonium was present in the medium. Since synthesis of new nitrate reductase should be inhibited in the presence of CM, nitrate would not be converted into useable nitrogen for the cell. Figure 4 also shows that when ammonium-grown cells were starved for nitrogen, the amount of nitrate reductase activity increased; starved cells transferred to nitrate with or without CM should therefore have the ability to utilize nitrate more quickly than nitrogen-replete cells, a finding consistent with our observations. (Compare Tables 1 and 2.) However, 15N was first observed in cyanophycin in ammonium/starve/nitrate experiments at the latest time (6 h) of any of the nitrogen starvation (without CM) experiments (Table 1). Since, as shown in Fig. 4, the activity of nitrate reductase increased almost 10-fold during ammonium starvation (derepression), nitrate reductase could not have been limiting in this case.
In all cases in which CM was added with 15N to washed, but non-nitrogen-starved cells grown first in 14N (Table 2), 15N was incorporated into cyanophycin more slowly than in starved cells (Table 1), suggesting that new protein synthesis is necessary for the new nitrogen source to be incorporated and/or that more cyanophycin is made from nitrogen derived from cellular protein than from extracellular nitrogen in the nonstarved cells. On the other hand, for nitrogen-starved cells, whether or not CM was added with the source of replenishment nitrogen (Table 1), there was little effect on the time and rate at which extracellular nitrogen is incorporated into cyanophycin except in the case of ammonium/starve/nitrate cells. These cells incorporated medium nitrogen into cyanophycin earlier, and to a greater extent, in the presence of CM than without CM. Protein synthesis, therefore, does not appear to be necessary for either nitrate or ammonium incorporation into cyanophycin for nitrogen starved cells, even when switching nitrogen sources.
The regulation of cyanophycin formation could take place at many points when cells are nitrogen stressed or transferred to media with different nitrogen sources. Many proteins in addition to nitrate reductase are involved in nitrogen uptake and assimilation and in cyanophycin synthesis and degradation. The levels of these proteins appear to depend on whether or not nitrogen is limited and on whether nitrogen is present in the form of ammonium (23). In excess nitrogen, cyanophycin synthesis is promoted (3), and PII is in its nonphosphorylated form (12, 18). Nonphosphorylated PII was recently shown to stimulate N-acetyl glutamate kinase, which favors arginine synthesis and, hence, cyanophycin synthesis (15). Thus, cyanophycin synthesis may be enhanced by the presence of several enzymes present under conditions of excess nitrogen.
In the absence of ammonium, the cph genes are more highly expressed, and more cyanophycin accumulated in both filaments and heterocysts of Anabaena sp. strain PCC 7120 (29). If the cphA gene is also expressed at high levels during nitrogen starvation in Synechocystis sp. strain PCC 6308, the cyanophycin synthetase produced would be available to synthesize cyanophycin when nitrogen is replenished. A lower level of expression of the cph genes in the presence of ammonium in Synechocystis could explain the late appearance of cyanophycin in washed nitrogen replete cells (Table 2). However, all of the data for the addition of ammonium or ammonium plus CM to nitrogen-starved cells (Table 1) suggest rapid synthesis of cyanophycin from medium nitrogen less than 1 h after ammonium is added. These data also confirm that protein synthesis is not necessary for cyanophycin synthesis and that cyanophycin is synthesized more rapidly in the presence of a protein synthesis inhibitor when protein synthesis is not competing for nitrogen.
The experiments described here suggest that nitrogen is incorporated into cyanophycin from both the breakdown of cellular protein and directly from the medium. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether or not the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells, but further work is necessary to determine whether and how other enzymes or transcription factors are involved. in Synechocystis sp. strain PCC 6308.
ACKNOWLEDGMENTS
This study was supported by NSF grants MCB 9728609, DBI 9732414, CHE 9601526, and OIA 9873771; a grant to Wellesley College from the Howard Hughes Medical Institute Undergraduate Science Education Program; and Wellesley College.
We gratefully acknowledge the scientific contributions of the following undergraduate students who participated in this work: Jiae Kim, Cristina Chae, Melissa Davis, Manisha Sijapati, and Salima Sheikh.
REFERENCES
Aldehni, M. F., J. Sauer, C. Spielhauper, R. Schmid, and K. Forchhammer. 2003. Signal transduction protein PII is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongates strain PCC 7941. J. Bacteriol. 185:2582-2591.
Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4.
Allen, M. M. 1984. Cyanobacterial cell inclusions. Annu. Rev. Microbiol. 38:1-25.
Allen, M. M., and F. Hutchison. 1980. Nitrogen limitation and recovery in the cyanobacterium Aphanocapsa 6308. Arch. Microbiol. 128:1-7.
Allen, M. M., F. Hutchison, and P. J. Weathers. 1980. Cyanophycin granule polypeptide (CGP) formation and degradation in the cyanobacterium Aphanocapsa 6308. J. Bacteriol. 141:687-693.
Allen, M. M., C. M. Yuen, L. Medeiros, N. Zizlsperger, M. Farooq, and N. H. Kolodny. 2005. Effects of light and chloramphenicol stress on incorporation of nitrogen into cyanophycin in Synechocystis sp. strain PCC 6308. Biochim. Biophys. Acta 1725:241-246.
Boussiba, S., and J. Gibson. 1991. Ammonia translocation in cyanobacteria. FEMS Microbiol. Rev. 88:1-14.
Carr, N. G. 1988. Nitrogen reserves and dynamic reservoirs in cyanobacteria, p. 13-39. In L. J. Rogers and J. R. Gallon (ed.), Biochemistry of the algae and cyanobacteria. Clarendon Press, Oxford, United Kingdom.
Erickson, N. A., N. H. Kolodny, and M. M. Allen. 2001. A rapid and sensitive method for the analysis of cyanophycin. Biochim. Biophys. Acta 1526:5-9.
Flores, E., and A. Herrero. 1994. Assimilatory nitrogen metabolism and its regulation, p. 487-517. In D. A. Bryant (ed.), The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Flores, E., and A. Herrero. 2005. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem. Soc. Trans. 33:164-167.
Forchhammer, K. 2004. Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol. Rev. 28:319-333.
Forchhammer, K., and N. Tandeau de Marsac. 1994. The PII 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.
Garcia-Dominguez, M., J. C. Reyes, and F. J. Florencio. 2000. NtcA represses transcription of gifA and gifB genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. strain 6803. Mol. Microbiol. 35:1192-1201.
Heinrich, A., M. Maheswaran, U. Ruppert, and K. Forchhammer. 2004. The Synechococcus elongates PII signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase. Mol. Microbiol. 52:1202-1314.
Herrero, A., E. Flores, and M. G. Guerrero. 1981. Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 6119, and Nostoc sp. strain 6717. J. Bacteriol. 145:175-180.
Herrero, A., A. M. Muro-Pastor, and E. Flores. 2001. Nitrogen control in cyanobacteria. J. Bacteriol. 183:411-425.
Lee, H.-M., E. Flores, A. Herrero, J. Houmard, and N. Tandeau de Marsac. 1998. A role for the signal transduction protein PII in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Lett. 427:292-295.
Leganes, F., F. Fernandes-Pinas, and C. P. Wolk. 1998. A transposition-induced mutant of Nostoc ellipsoporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinites. Microbiology 144:1799-1805.
Li, H., D. M. Sherman, S. Bao, and L. A. Sherman. 2001. Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria. Arch. Microbiol. 176:9-18.
Luque, I., Vazquez-Bermudez, M. F., J. Paz-Yepes, E. Flores, and A. Herrero. 2004. In vivo activity of the nitrogen control transcription factor NtcA is subjected to metabolic regulation in Synechococcus sp. strain PCC 7942. FEMS Microbiol. Lett. 236:47-52.
Mackerras, A. H., N. M. De Chazal, and G. D. Smith. 1990. Transient accumulations of cyanophycin in Anabaena cylindrica and Synechocystis 6803. J. Gen. Microbiol. 136:2057-2065.
Merida, A., P. Candau, and F. J. Florencio. 1991. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium. J. Bacteriol. 173:4095-4100.
Montesinos, M. L., A. M. Muro-Pastor, A. Herrero, and E. Flores. 1998. Ammonium/methylammonium permeases of a cyanobacterium. J. Biol. Chem. 273:31463-31470.
Muro-Pastor, M. 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.
Omata, T., M. Ohmori, N. Arai, and T. Ogawa. 1989. Genetically engineered mutant of the cyanobacterium Synecococcus PCC 7942 defective in nitrate transport. Proc. Natl. Acad. Sci. USA 86:6612-6616.
Omata, T., X. Andriesse, and A. Hirano. 1993. Identification and characterization of a gene involved in nitrate assimilation in the cyanobacterium Synechococcus sp. PCC 7942. Mol. Gen. Genet. 236:193-202.
Paz-Yepes, J., E. Flores, and A. Herrero. 2003. Transcriptional effects of the signal transduction protein PII (glnB gene product) on NtcA-dependent genes in Synechococcus sp. PCC 7942. FEBS Lett. 534:42-46.
Picossi, S., A. Valladares, E. Flores, and A. Herrero. 2004. Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer: expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. J. Biol. Chem. 279:11582-11592.
Saha, S. K., L. Uma, and G. Subramanian. 2003. Nitrogen stress induced changes in the marine cyanobacterium Oscillatoria willei BDU 130511. FEMS Microbiol. Ecol. 45:263-272.
Sherman, D. M., D. Tucker, and L. A. Sherman. 2000. Heterocyst development and localization of cyanophycin in nitrogen-fixing cultures of Anabaena sp. PCC 7120 (cyanobacteria). J. Phycol. 36:932-941.
Shibata, K. 1959. Spectrophotometry of translucent biological materials: opal glass transmission method, p. 77-109. In D. Glick (ed.), Methods of biochemical analysis. Interscience Press, Inc., New York, N.Y.
Simon, R. D. 1973. The effect of chloramphenicol on the production of cyanophycin granule polypeptide in the blue-green alga Anabaena cylindrica. Arch. Microbiol. 92:115-122.
Suarez, C., S. J. Kohler, M. M. Allen, and N. H. Kolodny. 1999. NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain 6308. Biochim. Biophys. Acta 1426:429-438.
Vazquez-Bermudez, M. F., A. Herrero, and E. Flores. 2003. Carbon supply and 2-oxoglutarate effects on expression of nitrate reductase and nitrogen-regulated genes in Synechococcus sp. strain PCC 7942. FEMS Microbiol. Lett. 221:155-159.
Ziegler, K., D. P. Stephan, E. K. Pistorius, H. G. Ruppel, and W. Lockau. 2001. A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects. FEMS Microbiol. Lett. 196:13-18.
Zuther, E., H. Schubert, and M. Hagemann. 1998. Mutation of a gene encoding a putative glycoprotease leads to reducted tolerance, altered pigmentation, and cyanophycin accumulation in the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 180:1715-1722.(Nancy H. Kolodny, Deborah)
ABSTRACT
Experiments were carried out to examine the effects of nitrogen source on nitrogen incorporation into cyanophycin during nitrogen limitation and repletion, both with or without inhibition of protein synthesis, in cyanobacteria grown on either nitrate or ammonium. The use of nitrate and ammonium, 14N labeled in the growth medium and 15N labeled in the repletion medium, allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance spectroscopy. The data suggest that nitrogen from both the breakdown of cellular protein (14N) and directly from the medium (15N) is incorporated into cyanophycin. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells and to the possible expression of cyanophycin synthetase during nitrogen starvation.
INTRODUCTION
Cyanobacteria are capable of growing on a number of different nitrogen sources. Nitrate, nitrite, ammonium, urea, several amino acids, atmospheric nitrogen, nucleosides, and bases all have been reported to be assimilated by some cyanobacteria, but ammonium appears to be assimilated preferentially (7, 17). Nitrate is probably the most abundant form of combined nitrogen available for assimilation by cyanobacteria (10). The nondiazotrophic cyanobacterium Synechocystis sp. strain PCC 6308 has been shown to metabolize nitrate, ammonium, and diamino acids (4) and to incorporate the nitrogen from these sources into necessary metabolites, as well as into the nitrogen storage material, cyanophycin granule polypeptide (cyanophycin). Nitrogen limitation in nondiazotrophic cyanobacteria leads to many different effects that have been described in a variety of strains (4, 30).
Utilization of different nitrogen sources first requires their passage through the permeability barrier of the cytoplasmic membrane into the cyanobacterial cell. At low nitrate concentrations, the endergonic uptake of nitrate in freshwater cyanobacteria takes place through ABC-type transporters that exhibit high affinity for nitrate (27). Passive diffusion of nitrate takes place in Synecchococcus sp. strain PCC 7942 at nitrate concentrations above 1 mM (26). Ammonium at low concentrations (<0.25 mM) is taken up by a permease that catalyzes a membrane potential-dependent active transport, but this system is repressed in higher concentrations of ammonium where passive diffusion of unprotonated ammonia may be the mechanism for net uptake of ammonium (24). After diffusion, intracellular ammonium is trapped by glutamine synthetase. All nitrogen sources that are assimilated are converted to intracellular ammonium. Intracellular nitrate, for example, is reduced to nitrite by nitrate reductase; the nitrite is then reduced to ammonium by nitrite reductase. The ammonium then enters the glutamine synthetase/glutamate synthase cycle to be incorporated into 2-oxoglutarate (21, 24, 25, 35).
Many forms of transcriptional and posttranslational control have been shown to be involved in nitrogen assimilation when cells are grown under different physiological conditions (reviewed in references 11 and 12). A global nitrogen-regulating gene, ntcA, has been shown to synthesize a transcriptional regulator whose activity is influenced by 2-oxoglutarate. This gene is activated by the ATP binding, signal transduction protein PII (glnB gene product [13]) especially when the C-to-N ratio in the cell is high (21, 28), as is true in cases of nitrogen limitation. PII in its phosphorylated form also is required for the activation of transcription of NtcA-dependent genes, such as nitrate reductase, under conditions of nitrogen limitation (1). However, when cells are placed in conditions where there are low 2-oxoglutarate levels (high N-to-C levels), the nonphosphorylated form of PII appears to cause inhibition of the nitrate/nitrite permease (18). In nitrogen-starved Synechocystis sp. strain PCC 6803, the expression of three ammonium permeases was higher than when cells were incubated in either ammonium or nitrate (24). Similarly, glutamine synthetase in Synechocystis sp. strain PCC 6803 is inactivated in the presence of ammonium by two small protein factors (IF7 and IF17); these factors are regulated by NtcA (14).
Two molecules, both with high nitrogen content, appear to be nitrogen storage compounds in cyanobacteria: phycocyanin and cyanophycin. Phycocyanin is the principal accessory pigment in most cyanobacteria, whereas cyanophycin is thought to function primarily as a nitrogen reserve. Cyanophycin, a non-ribosomally synthesized peptide, composed of arginine and aspartic acid, accumulates in cyanobacteria when they are grown under all unbalanced nutrient conditions except nitrogen starvation (33, 3). Cyanobacteria that synthesize cyanophycin tend to use it as a nitrogen source during nitrogen starvation before using phycocyanin and other sources of cellular nitrogen such as proteins (5).
There is evidence that cyanophycin acts as transient store for newly fixed nitrogen in the heterocysts of diazotrophic cyanobacteria (19), and several studies have suggested that cyanophycin is a dynamic reservoir in most cyanobacteria (8, 22, 20). When cyanobacteria are starved for nitrogen, cyanophycin is broken down; when nitrogen becomes available, cyanophycin is again synthesized. Upon reintroduction of nitrogen, cells rebuild their nitrogen stores and begin to grow. It appears that cyanophycin may play an even more important role as a nitrogen reserve in diazotrophic unicellular strains (20) than in nondiazotrophic unicellular strains such as Synechocystis sp. strain PCC 6803, where phycobilisomes appear to be the main nitrogen reserve. Picossi et al. (29) showed that expression of the genes for cyanophycin metabolism (cphA for cyanophycin synthesis and cphB for cyanophycinase) increases in the absence of ammonium in Anabaena sp. strain PCC 7120. The inability to degrade cyanophycin is detrimental to the diazotrophic growth of this strain.
Cyanophycin is synthesized in Synechocytis sp. strain PCC 6308 immediately after nitrogen is replenished (4), whereas phycocyanin is not synthesized until about 6 h later. The synthesis of cyanophycin peaks at up to 12% of the dry weight of the cells by 10 to 12 h and then decreases to the low levels found in typical exponential growth. After growth of the same strain in medium containing ammonium, and then starvation and replenishing with ammonium, Mackerras et al. (22) also found that cyanophycin was synthesized immediately and then degraded within 24 h. A salt-sensitive, glycoprotease-negative mutant (gcp mutant) of Synechocystis sp. strain PCC 6803 accumulated cyanophycin to a level 40 times that of the wild-type strain since it was unable to remobilize cyanophycin (37). In this mutant, phycobilisomes were degraded as a source of nitrogen.
Recently, we have shown that cells grown in low light with or without the protein synthesis inhibitor chloramphenicol (CM), as well as cells grown in normal light with CM, synthesize cyanophycin from nitrogen obtained both from degradation of cellular proteins and directly from the medium (6). Nitrogen from the medium was incorporated at a faster rate and to a greater extent than nitrogen from protein degradation.
Since cyanophycin is a nitrogen storage molecule that may be a dynamic reserve, the experiments described here were designed to determine how the source of nitrogen affects whether cyanophycin is synthesized from nitrogen derived from proteolysis of cellular protein or from nitrogen in the cells' growth medium. Experiments were carried out here to examine the effects of nitrogen limitation on nitrogen incorporation into cyanophycin with or without inhibition of protein synthesis in cells growing on either nitrate or ammonium. Cells were grown in 14N medium and then either washed or allowed to deplete their nitrogen before being transferred into 15N repletion medium. The use of 14N in the growth medium and 15N in the repletion medium allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance (NMR) spectroscopy (34). In all cases, the nitrogen in cyanophycin came from both the breakdown of cellular protein and nitrogen in the medium but at different rates and to different extents depending on the nitrogen source and stress condition.
MATERIALS AND METHODS
BG-11 medium (2) containing either 17.6 mM sodium nitrate (Na14NO3) or 10 mM ammonium chloride (14NH4Cl) and 2 mM TES (pH 10) was used for the growth and maintenance of cells. Experimental cells were grown at 35°C with cool white fluorescent light of approximately 80 μE s–1 m–2 in BG-11 medium containing 22.6 mM sodium carbonate in 1-liter Roux flasks that were continuously bubbled with 5% CO2 in air. Cells were starved for nitrogen by transferring exponentially growing cells to BG-11 medium containing the 14N forms of either 0.44 mM sodium nitrate or 1 mM ammonium chloride. Nitrogen starvation was observed by the culture's change in color from green to yellow, a process that usually required about 3 days. When starved, the culture was divided and added to new Roux flasks where cells were replenished with 10 mM 15NH4Cl (Cambridge Isotopes, Cambridge, MA) and 2 mM TES or with 5 to 17.6 mM Na15NO3 (Cambridge Isotopes). In some experiments, 5 μg of CM/ml was added at the same time as the nitrogen source. In experiments in which cultures were not nitrogen starved, cells were washed before the 15N source was added.
Cyanophycin was extracted from cells harvested immediately and at increasing times up to 24 h after transfer from starvation conditions or washing to 15N growth medium, either with or without CM, and dissolved in 0.1 M HCl as described by Allen et al. (6). 1H NMR spectra of all cyanophycin samples were run on a Bruker Avance spectrometer (Bruker Instruments, Billerica, MA) at 400.152 MHz (34, 6). Spectra were calibrated to a 0.010 M solution of TSP (3-trimethylsilyl-2,2,3,3-tetradeutero sodium propionate; Cambridge Isotopes) in D2O (Aldrich Chemical Co., Milwaukee, WI) and analyzed as described in Allen et al. (6), using the 1D WIN-NMR PC program (Bruker Instruments, Billerica, MA). In some cases only the NH peaks at 6.8 ppm (, '-14N, or 15N) and 7.4 ppm (-14N or 15N) were integrated because the small chemical shift difference between the -NH peaks for Asp and Arg (8.4 to 8.5 ppm) made the multiplets difficult to resolve and integrate accurately. Ratios of integration of peaks due to protons bonded to each 14N and 15N were divided by the sum of the integrations of the peaks for protons bonded to both nitrogen isotopes to calculate the percentages of each isotopic form of cyanophycin present at each time point. Total cyanophycin was determined by integrating the arginine -CH proton peak at 3.25 ppm (9). These integrations were standardized to the TSP integration and then corrected for the volume of the sample. Amounts of cyanophycin containing 14N and 15N at each time point were thus the product of the total cyanophycin and the fraction of each isotopic species.
Non-nitrogen-starved cells have different amounts of cyanophycin present when they are washed and placed into new nitrogen-containing medium, depending on their growth stage. Exponentially growing cells have small, but sometimes measurable amounts of cyanophycin when transferred. The amount of cyanophycin present in these cells (determined from the arginine -CH peak at 3.25 ppm) at the time of transfer was subtracted from the amount of cyanophycin at each point in time course experiments in order to calculate rates and times of incorporation of 14N and 15N comparable to the starved cell experiments. Without this correction, rates of 15N incorporation would have appeared lower and the time at which 15N=14N would have appeared later. Nitrogen-starved cells had no cyanophycin at the time of medium switch.
The concentration of chlorophyll a in the cultures was measured by adding 1 ml of methanol to 50 μl of culture, microfuging the mixture for 3 min, and measuring the absorbance of the supernatant at 665 nm. Pigments were also observed by recording whole-cell visible spectra between 400 and 750 nm using an opal glass technique (32). Nitrate reductase assays were performed at various times on cells grown with either nitrate or ammonium, with or without the addition of CM, as described by Herrero et al. (16). Cells were permeabilized by using toluene and incubated for 5 min at 30°C with substrate, methyl viologen, and sodium dithionite. The reaction was stopped and the colored nitrite complex developed by addition of sulfanilamide in HCl and N-(1-naphthyl)-ethylenediamine.
RESULTS
The quantitative data used to calculate the incorporation of nitrogen into the cyanophycin of Synechocytis sp. strain PCC 6308 under different stress conditions were derived from 1H NMR spectra of isolated cyanophycin solubilized in 0.1 M HCl. Figure 1 shows a typical series over time of 1H NMR spectra of cyanophycin extracted from cells grown in medium containing 14NH4+, starved for nitrogen, and then replenished in medium containing 15NH4+ and CM. All cyanophycin peaks are labeled. Note that with increasing time the peaks to the left of the large central water peak (protons bonded to nitrogens) became more complex, indicating the incorporation of 15N into the cyanophycin. Also note that these peaks and the peaks to the right of the water peak (protons bonded to carbons) increased in intensity with time, indicating production of cyanophycin. The size of the TSP peak was used to normalize all spectra. The large central peak is that of H2O, which, although suppressed by the NMR pulse sequence used, is still much larger than the peaks of cyanophycin.
Plots of the percentages of 14N and 15N cyanophycin for the same experiment are shown in Fig. 2. The graph shows data from the integration of all of the NH peaks for both nitrogen isotopes. The time at which the percentages of 14N and 15N cyanophycin became equal was between 0.5 and 1 h, showing that 15N was incorporated into cyanophycin very rapidly and that by 24 h 90% of the cyanophycin was labeled with medium nitrogen (15N). Using the percentages of the two nitrogen isotopes and the amount of cyanophycin at each time point, the rates of production of 14N and 15N cyanophycin were calculated and are shown in Fig. 3. The slopes of these lines give the relative rates of production for the two isotopic molecules, representing the amount of nitrogen in cyanophycin from breakdown of cellular protein (14N) or from the medium (15N). In this ammonium/starve/ammonium with CM experiment there was little incorporation of nitrogen from the breakdown of cellular protein during the 24 h of the experiment, whereas the rate of cyanophycin synthesis from medium nitrogen increased rapidly before 2 h. The ratio of rates of incorporation of 15N to 14N was 11:1.
The results of 1H NMR spectroscopy studies of the cyanophycin produced over time under 12 combinations of nitrogen source, stress, and replenishing conditions are shown in Tables 1 and 2. Each set of data was obtained from graphs similar to those in Fig. 1, 2, and 3. Table 1 is organized to systematically compare the effects on nitrogen-starved cells of CM and nitrogen source during growth and repletion. Table 2 compares the effects of nitrogen source on nitrogen-replete cells treated with CM. In half the cases shown in Table 1 the nitrogen source for replenishment remained the same as before starvation, while in the other half the source was switched.
Table 1 shows that the highest rates of incorporation of medium nitrogen into cyanophycin were observed in the ammonium/starve/ammonium experiments and the ammonium/starve/ammonium plus CM experiments where cells immediately took up ammonium from the medium and incorporated it into cyanophycin after replenishment. The ratio of rates of 15N:14N incorporation into cyanophycin were the highest in any of the 12 experiments at 19:1 and 11:1. The results in Table 1 for nitrogen-starved cells grown on ammonium and replenished with nitrate in the absence of CM show that cyanophycin is synthesized more slowly, incorporation of medium nitrogen (15N) occurs to a lesser extent, and the relative rates of incorporation of medium and protein nitrogen are lower when the nitrogen source is switched to nitrate after starvation than when it remains ammonium. For starved nitrate-grown cells replenished in the absence of CM the effects of nutrient switch are much smaller. When nitrogen-starved cells grown on ammonium were replenished with nitrate in the presence of CM the extent of medium nitrogen incorporation and the ratio of rates of incorporation of medium and protein nitrogen increased. CM had little effect when the nitrogen source was switched from nitrate to ammonium or was kept constant for nitrogen-starved cells.
Table 2 shows the response of nitrogen-replete cells to CM when the nitrogen source was the same or was switched. Nitrate-grown, nitrogen-replete cells utilized external nitrate or ammonium at approximately equal rates when treated with CM, but ammonium-grown nitrogen-replete cells utilized external ammonium slowly and external nitrate even more slowly when CM was present. For all of these nitrogen-replete cells treated with CM there was a lag before any new cyanophycin was synthesized, except in the case of cells grown initially in nitrate and replenished with nitrate.
Nitrate reductase activity was found to be significantly higher, by at least an order of magnitude, in cells growing in nitrate than in those growing in ammonium (t = 5.79, P = 3.99E-06). Figure 4 shows the activity of nitrate reductase expressed as μg of nitrite produced/μg of chlorophyll. Nitrate reductase activity was low in ammonium-grown cells but increased as ammonium was depleted from the medium during starvation. When starved cells were replenished with ammonium, enzyme activity decreased to its prestarvation level. When starved cells were replenished with nitrate, nitrate reductase activity increased dramatically until between 22 and 46 h after replenishing, after which activity began to decrease. When CM was added with nitrate at the time of replenishing nitrate-starved cells, nitrate reductase activity remained low for at least 5 h (data not shown), suggesting that the increase in nitrate reductase activity requires new protein synthesis.
DISCUSSION
Carr (8) first suggested that nitrogen might move in and out of cyanophycin during cell growth. It was later shown that cyanophycin constitutes a dynamic reservoir of fixed nitrogen in diazotrophic cyanobacteria (31). Using cyanophycin synthetase mutants, it was recently demonstrated that the path of newly synthesized organic nitrogen through cyanophycin in the heterocyst is not obligatory (36, 29). The purpose of the present experiments was to follow the path of nitrogen into cyanophycin when a nondiazotrophic cyanobacterium was grown in the presence of each of two of its common nitrogen sources: nitrate or ammonium.
Using 1H NMR spectroscopy of cyanophycin from cells grown first in medium with a 14N nitrogen source, followed by transfer into 15N medium, allows the quantification of amounts of nitrogen in the macromolecule from both sources. In these experiments, Synechocystis sp. strain PCC 6308, which forms large amounts of cyanophycin (5), was grown in either 14N nitrate or in 14N ammonium to exponential phase. Cells were then either starved for nitrogen or not starved. If starved, cells would have little or no cyanophycin and cellular proteins would be degraded. Cells that were not starved would have small, but varying amounts of cyanophycin and more 14N cellular protein. In the presence of CM, new protein synthesis would be prevented. New cyanophycin synthesis would thus have to come largely from the medium nitrogen (15N) directly, or from previously synthesized protein (14N), without going through newly synthesized proteins in the cell.
The results from these experiments suggest that nitrogen for cyanophycin synthesis comes preferentially from the external medium unless this nitrogen cannot be used due to either none being present or to the absence of the enzymes necessary for its utilization. Furthermore, since cyanophycin appears to be synthesized more slowly when the nitrogen source is switched after starvation than when it remains the same (Table 1), either new enzyme synthesis or enzyme activation is necessary. Nitrate reductase activity was very low when ammonium was present in the medium (Fig. 4), a finding in agreement with data for other strains of cyanobacteria (16). Thus, when nitrate was added to ammonium-grown cells without prior nitrogen starvation, low levels of nitrate reductase would be present and little nitrate could reach cyanophycin.
Lack of nitrate reductase activity in nitrogen-replete cells grown in ammonium medium before being transferred to nitrate and CM was hypothesized as the factor responsible for the very late and limited incorporation of medium nitrogen into cyanophycin (Table 2). Figure 4 shows that nitrate reductase activity was very low when ammonium was present in the medium. Since synthesis of new nitrate reductase should be inhibited in the presence of CM, nitrate would not be converted into useable nitrogen for the cell. Figure 4 also shows that when ammonium-grown cells were starved for nitrogen, the amount of nitrate reductase activity increased; starved cells transferred to nitrate with or without CM should therefore have the ability to utilize nitrate more quickly than nitrogen-replete cells, a finding consistent with our observations. (Compare Tables 1 and 2.) However, 15N was first observed in cyanophycin in ammonium/starve/nitrate experiments at the latest time (6 h) of any of the nitrogen starvation (without CM) experiments (Table 1). Since, as shown in Fig. 4, the activity of nitrate reductase increased almost 10-fold during ammonium starvation (derepression), nitrate reductase could not have been limiting in this case.
In all cases in which CM was added with 15N to washed, but non-nitrogen-starved cells grown first in 14N (Table 2), 15N was incorporated into cyanophycin more slowly than in starved cells (Table 1), suggesting that new protein synthesis is necessary for the new nitrogen source to be incorporated and/or that more cyanophycin is made from nitrogen derived from cellular protein than from extracellular nitrogen in the nonstarved cells. On the other hand, for nitrogen-starved cells, whether or not CM was added with the source of replenishment nitrogen (Table 1), there was little effect on the time and rate at which extracellular nitrogen is incorporated into cyanophycin except in the case of ammonium/starve/nitrate cells. These cells incorporated medium nitrogen into cyanophycin earlier, and to a greater extent, in the presence of CM than without CM. Protein synthesis, therefore, does not appear to be necessary for either nitrate or ammonium incorporation into cyanophycin for nitrogen starved cells, even when switching nitrogen sources.
The regulation of cyanophycin formation could take place at many points when cells are nitrogen stressed or transferred to media with different nitrogen sources. Many proteins in addition to nitrate reductase are involved in nitrogen uptake and assimilation and in cyanophycin synthesis and degradation. The levels of these proteins appear to depend on whether or not nitrogen is limited and on whether nitrogen is present in the form of ammonium (23). In excess nitrogen, cyanophycin synthesis is promoted (3), and PII is in its nonphosphorylated form (12, 18). Nonphosphorylated PII was recently shown to stimulate N-acetyl glutamate kinase, which favors arginine synthesis and, hence, cyanophycin synthesis (15). Thus, cyanophycin synthesis may be enhanced by the presence of several enzymes present under conditions of excess nitrogen.
In the absence of ammonium, the cph genes are more highly expressed, and more cyanophycin accumulated in both filaments and heterocysts of Anabaena sp. strain PCC 7120 (29). If the cphA gene is also expressed at high levels during nitrogen starvation in Synechocystis sp. strain PCC 6308, the cyanophycin synthetase produced would be available to synthesize cyanophycin when nitrogen is replenished. A lower level of expression of the cph genes in the presence of ammonium in Synechocystis could explain the late appearance of cyanophycin in washed nitrogen replete cells (Table 2). However, all of the data for the addition of ammonium or ammonium plus CM to nitrogen-starved cells (Table 1) suggest rapid synthesis of cyanophycin from medium nitrogen less than 1 h after ammonium is added. These data also confirm that protein synthesis is not necessary for cyanophycin synthesis and that cyanophycin is synthesized more rapidly in the presence of a protein synthesis inhibitor when protein synthesis is not competing for nitrogen.
The experiments described here suggest that nitrogen is incorporated into cyanophycin from both the breakdown of cellular protein and directly from the medium. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether or not the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells, but further work is necessary to determine whether and how other enzymes or transcription factors are involved. in Synechocystis sp. strain PCC 6308.
ACKNOWLEDGMENTS
This study was supported by NSF grants MCB 9728609, DBI 9732414, CHE 9601526, and OIA 9873771; a grant to Wellesley College from the Howard Hughes Medical Institute Undergraduate Science Education Program; and Wellesley College.
We gratefully acknowledge the scientific contributions of the following undergraduate students who participated in this work: Jiae Kim, Cristina Chae, Melissa Davis, Manisha Sijapati, and Salima Sheikh.
REFERENCES
Aldehni, M. F., J. Sauer, C. Spielhauper, R. Schmid, and K. Forchhammer. 2003. Signal transduction protein PII is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongates strain PCC 7941. J. Bacteriol. 185:2582-2591.
Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4.
Allen, M. M. 1984. Cyanobacterial cell inclusions. Annu. Rev. Microbiol. 38:1-25.
Allen, M. M., and F. Hutchison. 1980. Nitrogen limitation and recovery in the cyanobacterium Aphanocapsa 6308. Arch. Microbiol. 128:1-7.
Allen, M. M., F. Hutchison, and P. J. Weathers. 1980. Cyanophycin granule polypeptide (CGP) formation and degradation in the cyanobacterium Aphanocapsa 6308. J. Bacteriol. 141:687-693.
Allen, M. M., C. M. Yuen, L. Medeiros, N. Zizlsperger, M. Farooq, and N. H. Kolodny. 2005. Effects of light and chloramphenicol stress on incorporation of nitrogen into cyanophycin in Synechocystis sp. strain PCC 6308. Biochim. Biophys. Acta 1725:241-246.
Boussiba, S., and J. Gibson. 1991. Ammonia translocation in cyanobacteria. FEMS Microbiol. Rev. 88:1-14.
Carr, N. G. 1988. Nitrogen reserves and dynamic reservoirs in cyanobacteria, p. 13-39. In L. J. Rogers and J. R. Gallon (ed.), Biochemistry of the algae and cyanobacteria. Clarendon Press, Oxford, United Kingdom.
Erickson, N. A., N. H. Kolodny, and M. M. Allen. 2001. A rapid and sensitive method for the analysis of cyanophycin. Biochim. Biophys. Acta 1526:5-9.
Flores, E., and A. Herrero. 1994. Assimilatory nitrogen metabolism and its regulation, p. 487-517. In D. A. Bryant (ed.), The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Flores, E., and A. Herrero. 2005. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem. Soc. Trans. 33:164-167.
Forchhammer, K. 2004. Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol. Rev. 28:319-333.
Forchhammer, K., and N. Tandeau de Marsac. 1994. The PII 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.
Garcia-Dominguez, M., J. C. Reyes, and F. J. Florencio. 2000. NtcA represses transcription of gifA and gifB genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. strain 6803. Mol. Microbiol. 35:1192-1201.
Heinrich, A., M. Maheswaran, U. Ruppert, and K. Forchhammer. 2004. The Synechococcus elongates PII signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase. Mol. Microbiol. 52:1202-1314.
Herrero, A., E. Flores, and M. G. Guerrero. 1981. Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 6119, and Nostoc sp. strain 6717. J. Bacteriol. 145:175-180.
Herrero, A., A. M. Muro-Pastor, and E. Flores. 2001. Nitrogen control in cyanobacteria. J. Bacteriol. 183:411-425.
Lee, H.-M., E. Flores, A. Herrero, J. Houmard, and N. Tandeau de Marsac. 1998. A role for the signal transduction protein PII in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Lett. 427:292-295.
Leganes, F., F. Fernandes-Pinas, and C. P. Wolk. 1998. A transposition-induced mutant of Nostoc ellipsoporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinites. Microbiology 144:1799-1805.
Li, H., D. M. Sherman, S. Bao, and L. A. Sherman. 2001. Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria. Arch. Microbiol. 176:9-18.
Luque, I., Vazquez-Bermudez, M. F., J. Paz-Yepes, E. Flores, and A. Herrero. 2004. In vivo activity of the nitrogen control transcription factor NtcA is subjected to metabolic regulation in Synechococcus sp. strain PCC 7942. FEMS Microbiol. Lett. 236:47-52.
Mackerras, A. H., N. M. De Chazal, and G. D. Smith. 1990. Transient accumulations of cyanophycin in Anabaena cylindrica and Synechocystis 6803. J. Gen. Microbiol. 136:2057-2065.
Merida, A., P. Candau, and F. J. Florencio. 1991. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium. J. Bacteriol. 173:4095-4100.
Montesinos, M. L., A. M. Muro-Pastor, A. Herrero, and E. Flores. 1998. Ammonium/methylammonium permeases of a cyanobacterium. J. Biol. Chem. 273:31463-31470.
Muro-Pastor, M. 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.
Omata, T., M. Ohmori, N. Arai, and T. Ogawa. 1989. Genetically engineered mutant of the cyanobacterium Synecococcus PCC 7942 defective in nitrate transport. Proc. Natl. Acad. Sci. USA 86:6612-6616.
Omata, T., X. Andriesse, and A. Hirano. 1993. Identification and characterization of a gene involved in nitrate assimilation in the cyanobacterium Synechococcus sp. PCC 7942. Mol. Gen. Genet. 236:193-202.
Paz-Yepes, J., E. Flores, and A. Herrero. 2003. Transcriptional effects of the signal transduction protein PII (glnB gene product) on NtcA-dependent genes in Synechococcus sp. PCC 7942. FEBS Lett. 534:42-46.
Picossi, S., A. Valladares, E. Flores, and A. Herrero. 2004. Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer: expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. J. Biol. Chem. 279:11582-11592.
Saha, S. K., L. Uma, and G. Subramanian. 2003. Nitrogen stress induced changes in the marine cyanobacterium Oscillatoria willei BDU 130511. FEMS Microbiol. Ecol. 45:263-272.
Sherman, D. M., D. Tucker, and L. A. Sherman. 2000. Heterocyst development and localization of cyanophycin in nitrogen-fixing cultures of Anabaena sp. PCC 7120 (cyanobacteria). J. Phycol. 36:932-941.
Shibata, K. 1959. Spectrophotometry of translucent biological materials: opal glass transmission method, p. 77-109. In D. Glick (ed.), Methods of biochemical analysis. Interscience Press, Inc., New York, N.Y.
Simon, R. D. 1973. The effect of chloramphenicol on the production of cyanophycin granule polypeptide in the blue-green alga Anabaena cylindrica. Arch. Microbiol. 92:115-122.
Suarez, C., S. J. Kohler, M. M. Allen, and N. H. Kolodny. 1999. NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain 6308. Biochim. Biophys. Acta 1426:429-438.
Vazquez-Bermudez, M. F., A. Herrero, and E. Flores. 2003. Carbon supply and 2-oxoglutarate effects on expression of nitrate reductase and nitrogen-regulated genes in Synechococcus sp. strain PCC 7942. FEMS Microbiol. Lett. 221:155-159.
Ziegler, K., D. P. Stephan, E. K. Pistorius, H. G. Ruppel, and W. Lockau. 2001. A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects. FEMS Microbiol. Lett. 196:13-18.
Zuther, E., H. Schubert, and M. Hagemann. 1998. Mutation of a gene encoding a putative glycoprotease leads to reducted tolerance, altered pigmentation, and cyanophycin accumulation in the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 180:1715-1722.(Nancy H. Kolodny, Deborah)