Effect of Nitrogen Source on Cyanophycin Synthesis in Synechocystis sp. Strain PCC 6308

Experiments were carried out to examine the effects of nitrogensource on nitrogen incorporation into cyanophycin during nitrogenlimitation and repletion, both with or without inhibition ofprotein synthesis, in cyanobacteria grown on either nitrateor ammonium. The use of nitrate and ammonium, ¹⁴N labeled inthe growth medium and ¹⁵N labeled in the repletion medium, allowsthe determination of the source of nitrogen in cyanophycin usingproton nuclear magnetic resonance spectroscopy. The data suggestthat 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 ratesand to different extents, depending on the source of nitrogen(ammonium or nitrate) and whether the cells are first starvedfor nitrogen. These differences appear to be related to theactivity of nitrate reductase in cells and to the possible expressionof cyanophycin synthetase during nitrogen starvation.

INTRODUCTION

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Introduction

Cyanobacteria are capable of growing on a number of differentnitrogen sources. Nitrate, nitrite, ammonium, urea, severalamino acids, atmospheric nitrogen, nucleosides, and bases allhave 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 nitrogenavailable for assimilation by cyanobacteria (10). The nondiazotrophiccyanobacterium Synechocystis sp. strain PCC 6308 has been shownto metabolize nitrate, ammonium, and diamino acids (4) and toincorporate the nitrogen from these sources into necessary metabolites,as well as into the nitrogen storage material, cyanophycin granulepolypeptide (cyanophycin). Nitrogen limitation in nondiazotrophiccyanobacteria leads to many different effects that have beendescribed in a variety of strains (4, 30).

Utilization of different nitrogen sources first requires theirpassage through the permeability barrier of the cytoplasmicmembrane into the cyanobacterial cell. At low nitrate concentrations,the endergonic uptake of nitrate in freshwater cyanobacteriatakes place through ABC-type transporters that exhibit highaffinity for nitrate (27). Passive diffusion of nitrate takesplace in Synecchococcus sp. strain PCC 7942 at nitrate concentrationsabove 1 mM (26). Ammonium at low concentrations (<0.25 mM)is taken up by a permease that catalyzes a membrane potential-dependentactive transport, but this system is repressed in higher concentrationsof ammonium where passive diffusion of unprotonated ammoniamay be the mechanism for net uptake of ammonium (24). Afterdiffusion, intracellular ammonium is trapped by glutamine synthetase.All nitrogen sources that are assimilated are converted to intracellularammonium. Intracellular nitrate, for example, is reduced tonitrite by nitrate reductase; the nitrite is then reduced toammonium by nitrite reductase. The ammonium then enters theglutamine synthetase/glutamate synthase cycle to be incorporatedinto 2-oxoglutarate (21, 24, 25, 35).

Many forms of transcriptional and posttranslational controlhave been shown to be involved in nitrogen assimilation whencells are grown under different physiological conditions (reviewedin references 11 and 12). A global nitrogen-regulating gene,ntcA, has been shown to synthesize a transcriptional regulatorwhose activity is influenced by 2-oxoglutarate. This gene isactivated by the ATP binding, signal transduction protein P_II(glnB gene product [13]) especially when the C-to-N ratio inthe cell is high (21, 28), as is true in cases of nitrogen limitation.P_II in its phosphorylated form also is required for the activationof transcription of NtcA-dependent genes, such as nitrate reductase,under conditions of nitrogen limitation (1). However, when cellsare placed in conditions where there are low 2-oxoglutaratelevels (high N-to-C levels), the nonphosphorylated form of P_IIappears 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 whencells were incubated in either ammonium or nitrate (24). Similarly,glutamine synthetase in Synechocystis sp. strain PCC 6803 isinactivated in the presence of ammonium by two small proteinfactors (IF7 and IF17); these factors are regulated by NtcA(14).

Two molecules, both with high nitrogen content, appear to benitrogen storage compounds in cyanobacteria: phycocyanin andcyanophycin. Phycocyanin is the principal accessory pigmentin most cyanobacteria, whereas cyanophycin is thought to functionprimarily as a nitrogen reserve. Cyanophycin, a non-ribosomallysynthesized peptide, composed of arginine and aspartic acid,accumulates in cyanobacteria when they are grown under all unbalancednutrient conditions except nitrogen starvation (33, 3). Cyanobacteriathat synthesize cyanophycin tend to use it as a nitrogen sourceduring nitrogen starvation before using phycocyanin and othersources of cellular nitrogen such as proteins (5).

There is evidence that cyanophycin acts as transient store fornewly fixed nitrogen in the heterocysts of diazotrophic cyanobacteria(19), and several studies have suggested that cyanophycin isa dynamic reservoir in most cyanobacteria (8, 22, 20). Whencyanobacteria are starved for nitrogen, cyanophycin is brokendown; when nitrogen becomes available, cyanophycin is againsynthesized. Upon reintroduction of nitrogen, cells rebuildtheir nitrogen stores and begin to grow. It appears that cyanophycinmay play an even more important role as a nitrogen reserve indiazotrophic unicellular strains (20) than in nondiazotrophicunicellular 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 forcyanophycin metabolism (cphA for cyanophycin synthesis and cphBfor cyanophycinase) increases in the absence of ammonium inAnabaena sp. strain PCC 7120. The inability to degrade cyanophycinis detrimental to the diazotrophic growth of this strain.

Cyanophycin is synthesized in Synechocytis sp. strain PCC 6308immediately after nitrogen is replenished (4), whereas phycocyaninis not synthesized until about 6 h later. The synthesis of cyanophycinpeaks at up to 12% of the dry weight of the cells by 10 to 12h and then decreases to the low levels found in typical exponentialgrowth. After growth of the same strain in medium containingammonium, and then starvation and replenishing with ammonium,Mackerras et al. (22) also found that cyanophycin was synthesizedimmediately and then degraded within 24 h. A salt-sensitive,glycoprotease-negative mutant (gcp mutant) of Synechocystissp. strain PCC 6803 accumulated cyanophycin to a level 40 timesthat of the wild-type strain since it was unable to remobilizecyanophycin (37). In this mutant, phycobilisomes were degradedas a source of nitrogen.

Recently, we have shown that cells grown in low light with orwithout the protein synthesis inhibitor chloramphenicol (CM),as well as cells grown in normal light with CM, synthesize cyanophycinfrom nitrogen obtained both from degradation of cellular proteinsand directly from the medium (6). Nitrogen from the medium wasincorporated at a faster rate and to a greater extent than nitrogenfrom protein degradation.

Since cyanophycin is a nitrogen storage molecule that may bea dynamic reserve, the experiments described here were designedto determine how the source of nitrogen affects whether cyanophycinis synthesized from nitrogen derived from proteolysis of cellularprotein or from nitrogen in the cells' growth medium. Experimentswere carried out here to examine the effects of nitrogen limitationon nitrogen incorporation into cyanophycin with or without inhibitionof protein synthesis in cells growing on either nitrate or ammonium.Cells were grown in ¹⁴N medium and then either washed or allowedto deplete their nitrogen before being transferred into ¹⁵Nrepletion medium. The use of ¹⁴N in the growth medium and ¹⁵Nin the repletion medium allows the determination of the sourceof nitrogen in cyanophycin using proton nuclear magnetic resonance(NMR) spectroscopy (34). In all cases, the nitrogen in cyanophycincame from both the breakdown of cellular protein and nitrogenin the medium but at different rates and to different extentsdepending on the nitrogen source and stress condition.

MATERIALS AND METHODS

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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) wasused for the growth and maintenance of cells. Experimental cellswere grown at 35°C with cool white fluorescent light ofapproximately 80 µE s^–1 m^–2 in BG-11 mediumcontaining 22.6 mM sodium carbonate in 1-liter Roux flasks thatwere continuously bubbled with 5% CO₂ in air. Cells were starvedfor nitrogen by transferring exponentially growing cells toBG-11 medium containing the ¹⁴N forms of either 0.44 mM sodiumnitrate or 1 mM ammonium chloride. Nitrogen starvation was observedby the culture's change in color from green to yellow, a processthat usually required about 3 days. When starved, the culturewas divided and added to new Roux flasks where cells were replenishedwith 10 mM ¹⁵NH₄Cl (Cambridge Isotopes, Cambridge, MA) and 2mM TES or with 5 to 17.6 mM Na¹⁵NO₃ (Cambridge Isotopes). Insome experiments, 5 µg of CM/ml was added at the sametime as the nitrogen source. In experiments in which cultureswere not nitrogen starved, cells were washed before the ¹⁵Nsource was added.

Cyanophycin was extracted from cells harvested immediately andat increasing times up to 24 h after transfer from starvationconditions or washing to ¹⁵N growth medium, either with or withoutCM, and dissolved in 0.1 M HCl as described by Allen et al.(6). ¹H NMR spectra of all cyanophycin samples were run on aBruker Avance spectrometer (Bruker Instruments, Billerica, MA)at 400.152 MHz (34, 6). Spectra were calibrated to a 0.010 Msolution of TSP (3-trimethylsilyl-2,2,3,3-tetradeutero sodiumpropionate; 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 ( ${omega}$ , ${omega}$ '-¹⁴N, or¹⁵N) and 7.4 ppm ( ${delta}$ -¹⁴N or ¹⁵N) were integrated because the smallchemical shift difference between the ${alpha}$ -NH peaks for Asp andArg (8.4 to 8.5 ppm) made the multiplets difficult to resolveand integrate accurately. Ratios of integration of peaks dueto protons bonded to each ¹⁴N and ¹⁵N were divided by the sumof the integrations of the peaks for protons bonded to bothnitrogen isotopes to calculate the percentages of each isotopicform of cyanophycin present at each time point. Total cyanophycinwas determined by integrating the arginine ${delta}$ -CH proton peak at3.25 ppm (9). These integrations were standardized to the TSPintegration and then corrected for the volume of the sample.Amounts of cyanophycin containing ¹⁴N and ¹⁵N at each time pointwere thus the product of the total cyanophycin and the fractionof each isotopic species.

Non-nitrogen-starved cells have different amounts of cyanophycinpresent when they are washed and placed into new nitrogen-containingmedium, depending on their growth stage. Exponentially growingcells have small, but sometimes measurable amounts of cyanophycinwhen transferred. The amount of cyanophycin present in thesecells (determined from the arginine ${delta}$ -CH peak at 3.25 ppm) atthe time of transfer was subtracted from the amount of cyanophycinat each point in time course experiments in order to calculaterates and times of incorporation of ¹⁴N and ¹⁵N comparable tothe starved cell experiments. Without this correction, ratesof ¹⁵N incorporation would have appeared lower and the timeat which ¹⁵N=¹⁴N would have appeared later. Nitrogen-starvedcells had no cyanophycin at the time of medium switch.

The concentration of chlorophyll a in the cultures was measuredby adding 1 ml of methanol to 50 µl of culture, microfugingthe mixture for 3 min, and measuring the absorbance of the supernatantat 665 nm. Pigments were also observed by recording whole-cellvisible spectra between 400 and 750 nm using an opal glass technique(32). Nitrate reductase assays were performed at various timeson cells grown with either nitrate or ammonium, with or withoutthe addition of CM, as described by Herrero et al. (16). Cellswere permeabilized by using toluene and incubated for 5 minat 30°C with substrate, methyl viologen, and sodium dithionite.The reaction was stopped and the colored nitrite complex developedby addition of sulfanilamide in HCl and N-(1-naphthyl)-ethylenediamine.

RESULTS

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Results

The quantitative data used to calculate the incorporation ofnitrogen into the cyanophycin of Synechocytis sp. strain PCC6308 under different stress conditions were derived from ¹HNMR spectra of isolated cyanophycin solubilized in 0.1 M HCl.Figure 1 shows a typical series over time of ¹H NMR spectraof cyanophycin extracted from cells grown in medium containing¹⁴NH₄⁺, starved for nitrogen, and then replenished in mediumcontaining ¹⁵NH₄⁺ and CM. All cyanophycin peaks are labeled.Note that with increasing time the peaks to the left of thelarge central water peak (protons bonded to nitrogens) becamemore complex, indicating the incorporation of ¹⁵N into the cyanophycin.Also note that these peaks and the peaks to the right of thewater peak (protons bonded to carbons) increased in intensitywith time, indicating production of cyanophycin. The size ofthe TSP peak was used to normalize all spectra. The large centralpeak is that of H₂O, which, although suppressed by the NMR pulsesequence used, is still much larger than the peaks of cyanophycin.

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FIG. 1. ¹H NMR spectra of cyanophycin over time extracted from cells grown on ¹⁴NH₄⁺, starved, and replenished with ¹⁵NH₄⁺ and CM at 0 min. A broad water peak at 0 min demonstrates the effect of poor water suppression. Peaks to the left of the water peak are protons bonded to nitrogen, and those to the right of the water peak are protons bonded to carbon.

Plots of the percentages of ¹⁴N and ¹⁵N cyanophycin for thesame experiment are shown in Fig. 2. The graph shows data fromthe integration of all of the NH peaks for both nitrogen isotopes.The time at which the percentages of ¹⁴N and ¹⁵N cyanophycinbecame equal was between 0.5 and 1 h, showing that ¹⁵N was incorporatedinto cyanophycin very rapidly and that by 24 h 90% of the cyanophycinwas labeled with medium nitrogen (¹⁵N). Using the percentagesof the two nitrogen isotopes and the amount of cyanophycin ateach time point, the rates of production of ¹⁴N and ¹⁵N cyanophycinwere calculated and are shown in Fig. 3. The slopes of theselines give the relative rates of production for the two isotopicmolecules, representing the amount of nitrogen in cyanophycinfrom breakdown of cellular protein (¹⁴N) or from the medium(¹⁵N). In this ammonium/starve/ammonium with CM experiment therewas little incorporation of nitrogen from the breakdown of cellularprotein during the 24 h of the experiment, whereas the rateof cyanophycin synthesis from medium nitrogen increased rapidlybefore 2 h. The ratio of rates of incorporation of ¹⁵N to ¹⁴Nwas 11:1.

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FIG. 2. Evolution of percent ¹⁴N and percent ¹⁵N over time in cells grown on ¹⁴NH₄⁺, starved, and replenished with ¹⁵NH₄⁺ and CM. Solid symbols, ¹⁴N; open symbols, ¹⁵N [ ${circ}$ , ${omega}$ , ${omega}$ '(Arg); ${square}$ , ${delta}$ (Arg); ${diamond}$ , amide ( ${alpha}$ )(Asp, Arg)]. Ratios of integrations 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.

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FIG. 3. Amount of incorporation of ¹⁴N and ¹⁵N (arbitrary units) into cyanophycin for cells grown on ¹⁴NH₄⁺, starved, and replenished with ¹⁵NH₄⁺ and CM plotted versus time. Amounts of cyanophycin containing ¹⁴N and ¹⁵N at each time point are the product of the total cyanophycin and the fraction of each isotopic species. ${blacksquare}$ , ¹⁵N; ${blacklozenge}$ , ¹⁴N. The slopes of these lines are the rates of incorporation of ¹⁴N and ¹⁵N into cyanophycin.

The results of ¹H NMR spectroscopy studies of the cyanophycinproduced over time under 12 combinations of nitrogen source,stress, and replenishing conditions are shown in Tables 1 and2. Each set of data was obtained from graphs similar to thosein Fig. 1, 2, and 3. Table 1 is organized to systematicallycompare the effects on nitrogen-starved cells of CM and nitrogensource during growth and repletion. Table 2 compares the effectsof nitrogen source on nitrogen-replete cells treated with CM.In half the cases shown in Table 1 the nitrogen source for replenishmentremained the same as before starvation, while in the other halfthe source was switched.

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TABLE 1. Comparison of effect of CM on cyanophycin synthesis in nitrogen-starved cells grown and replenished with either nitrate or ammonium

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TABLE 2. Comparison of effects of nitrogen sources on cyanophycin synthesis in nitrogen-replete cells treated with CM

Table 1 shows that the highest rates of incorporation of mediumnitrogen into cyanophycin were observed in the ammonium/starve/ammoniumexperiments and the ammonium/starve/ammonium plus CM experimentswhere cells immediately took up ammonium from the medium andincorporated it into cyanophycin after replenishment. The ratioof rates of ¹⁵N:¹⁴N incorporation into cyanophycin were thehighest in any of the 12 experiments at 19:1 and 11:1. The resultsin Table 1 for nitrogen-starved cells grown on ammonium andreplenished with nitrate in the absence of CM show that cyanophycinis synthesized more slowly, incorporation of medium nitrogen(¹⁵N) occurs to a lesser extent, and the relative rates of incorporationof medium and protein nitrogen are lower when the nitrogen sourceis switched to nitrate after starvation than when it remainsammonium. For starved nitrate-grown cells replenished in theabsence of CM the effects of nutrient switch are much smaller.When nitrogen-starved cells grown on ammonium were replenishedwith nitrate in the presence of CM the extent of medium nitrogenincorporation and the ratio of rates of incorporation of mediumand protein nitrogen increased. CM had little effect when thenitrogen source was switched from nitrate to ammonium or waskept constant for nitrogen-starved cells.

Table 2 shows the response of nitrogen-replete cells to CM whenthe nitrogen source was the same or was switched. Nitrate-grown,nitrogen-replete cells utilized external nitrate or ammoniumat approximately equal rates when treated with CM, but ammonium-grownnitrogen-replete cells utilized external ammonium slowly andexternal nitrate even more slowly when CM was present. For allof these nitrogen-replete cells treated with CM there was alag before any new cyanophycin was synthesized, except in thecase of cells grown initially in nitrate and replenished withnitrate.

Nitrate reductase activity was found to be significantly higher,by at least an order of magnitude, in cells growing in nitratethan in those growing in ammonium (t = 5.79, P = 3.99E-06).Figure 4 shows the activity of nitrate reductase expressed asµg of nitrite produced/µg of chlorophyll. Nitratereductase activity was low in ammonium-grown cells but increasedas ammonium was depleted from the medium during starvation.When starved cells were replenished with ammonium, enzyme activitydecreased to its prestarvation level. When starved cells werereplenished with nitrate, nitrate reductase activity increaseddramatically until between 22 and 46 h after replenishing, afterwhich activity began to decrease. When CM was added with nitrateat the time of replenishing nitrate-starved cells, nitrate reductaseactivity remained low for at least 5 h (data not shown), suggestingthat the increase in nitrate reductase activity requires newprotein synthesis.

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FIG. 4. Nitrate reductase activity during starvation of ammonium-grown cells in ammonium starvation medium followed by replenishment of these cells with ammonium or nitrate. Activity is expressed in terms of µg of nitrite/µg of chlorophyll. Nitrogen starvation began at time zero and cells were replenished with nitrogen at 96 h. Error bars represent range of data for three trials at each time point. ( ${blacklozenge}$ , ammonium starvation. ${blacksquare}$ , replenished with ammonium; ${blacktriangleup}$ , replenished with nitrate.

DISCUSSION

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Discussion

Carr (8) first suggested that nitrogen might move in and outof cyanophycin during cell growth. It was later shown that cyanophycinconstitutes a dynamic reservoir of fixed nitrogen in diazotrophiccyanobacteria (31). Using cyanophycin synthetase mutants, itwas recently demonstrated that the path of newly synthesizedorganic nitrogen through cyanophycin in the heterocyst is notobligatory (36, 29). The purpose of the present experimentswas to follow the path of nitrogen into cyanophycin when a nondiazotrophiccyanobacterium was grown in the presence of each of two of itscommon nitrogen sources: nitrate or ammonium.

Using ¹H NMR spectroscopy of cyanophycin from cells grown firstin medium with a ¹⁴N nitrogen source, followed by transfer into¹⁵N medium, allows the quantification of amounts of nitrogenin the macromolecule from both sources. In these experiments,Synechocystis sp. strain PCC 6308, which forms large amountsof cyanophycin (5), was grown in either ¹⁴N nitrate or in ¹⁴Nammonium to exponential phase. Cells were then either starvedfor nitrogen or not starved. If starved, cells would have littleor no cyanophycin and cellular proteins would be degraded. Cellsthat were not starved would have small, but varying amountsof cyanophycin and more ¹⁴N cellular protein. In the presenceof CM, new protein synthesis would be prevented. New cyanophycinsynthesis 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 forcyanophycin synthesis comes preferentially from the externalmedium unless this nitrogen cannot be used due to either nonebeing present or to the absence of the enzymes necessary forits utilization. Furthermore, since cyanophycin appears to besynthesized more slowly when the nitrogen source is switchedafter starvation than when it remains the same (Table 1), eithernew enzyme synthesis or enzyme activation is necessary. Nitratereductase activity was very low when ammonium was present inthe medium (Fig. 4), a finding in agreement with data for otherstrains of cyanobacteria (16). Thus, when nitrate was addedto ammonium-grown cells without prior nitrogen starvation, lowlevels of nitrate reductase would be present and little nitratecould reach cyanophycin.

Lack of nitrate reductase activity in nitrogen-replete cellsgrown in ammonium medium before being transferred to nitrateand CM was hypothesized as the factor responsible for the verylate and limited incorporation of medium nitrogen into cyanophycin(Table 2). Figure 4 shows that nitrate reductase activity wasvery low when ammonium was present in the medium. Since synthesisof new nitrate reductase should be inhibited in the presenceof CM, nitrate would not be converted into useable nitrogenfor the cell. Figure 4 also shows that when ammonium-grown cellswere starved for nitrogen, the amount of nitrate reductase activityincreased; starved cells transferred to nitrate with or withoutCM should therefore have the ability to utilize nitrate morequickly than nitrogen-replete cells, a finding consistent withour observations. (Compare Tables 1 and 2.) However, ¹⁵N wasfirst observed in cyanophycin in ammonium/starve/nitrate experimentsat the latest time (6 h) of any of the nitrogen starvation (withoutCM) experiments (Table 1). Since, as shown in Fig. 4, the activityof nitrate reductase increased almost 10-fold during ammoniumstarvation (derepression), nitrate reductase could not havebeen limiting in this case.

In all cases in which CM was added with ¹⁵N to washed, but non-nitrogen-starvedcells grown first in ¹⁴N (Table 2), ¹⁵N was incorporated intocyanophycin more slowly than in starved cells (Table 1), suggestingthat new protein synthesis is necessary for the new nitrogensource to be incorporated and/or that more cyanophycin is madefrom nitrogen derived from cellular protein than from extracellularnitrogen in the nonstarved cells. On the other hand, for nitrogen-starvedcells, whether or not CM was added with the source of replenishmentnitrogen (Table 1), there was little effect on the time andrate at which extracellular nitrogen is incorporated into cyanophycinexcept in the case of ammonium/starve/nitrate cells. These cellsincorporated medium nitrogen into cyanophycin earlier, and toa greater extent, in the presence of CM than without CM. Proteinsynthesis, therefore, does not appear to be necessary for eithernitrate or ammonium incorporation into cyanophycin for nitrogenstarved cells, even when switching nitrogen sources.

The regulation of cyanophycin formation could take place atmany points when cells are nitrogen stressed or transferredto media with different nitrogen sources. Many proteins in additionto nitrate reductase are involved in nitrogen uptake and assimilationand in cyanophycin synthesis and degradation. The levels ofthese proteins appear to depend on whether or not nitrogen islimited and on whether nitrogen is present in the form of ammonium(23). In excess nitrogen, cyanophycin synthesis is promoted(3), and P_II is in its nonphosphorylated form (12, 18). NonphosphorylatedP_II 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 presenceof 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 heterocystsof Anabaena sp. strain PCC 7120 (29). If the cphA gene is alsoexpressed at high levels during nitrogen starvation in Synechocystissp. strain PCC 6308, the cyanophycin synthetase produced wouldbe available to synthesize cyanophycin when nitrogen is replenished.A lower level of expression of the cph genes in the presenceof ammonium in Synechocystis could explain the late appearanceof cyanophycin in washed nitrogen replete cells (Table 2). However,all of the data for the addition of ammonium or ammonium plusCM to nitrogen-starved cells (Table 1) suggest rapid synthesisof cyanophycin from medium nitrogen less than 1 h after ammoniumis added. These data also confirm that protein synthesis isnot necessary for cyanophycin synthesis and that cyanophycinis synthesized more rapidly in the presence of a protein synthesisinhibitor when protein synthesis is not competing for nitrogen.

The experiments described here suggest that nitrogen is incorporatedinto cyanophycin from both the breakdown of cellular proteinand directly from the medium. Nitrogen is incorporated intocyanophycin at different rates and to different extents, dependingon the source of nitrogen (ammonium or nitrate) and whetheror not the cells are first starved for nitrogen. These differencesappear to be related to the activity of nitrate reductase incells, but further work is necessary to determine whether andhow other enzymes or transcription factors are involved. inSynechocystis 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 fromthe Howard Hughes Medical Institute Undergraduate Science EducationProgram; and Wellesley College.

We gratefully acknowledge the scientific contributions of thefollowing undergraduate students who participated in this work:Jiae Kim, Cristina Chae, Melissa Davis, Manisha Sijapati, andSalima Sheikh.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, Wellesley College, 106 Central St., Wellesley, MA 02481. Phone: (781) 283-3068. Fax: (781) 283-3642. E-mail: mallen{at}wellesley.edu.

REFERENCES

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