Vol. 140, No. 2

JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 508-517

0021-9193/79/11-0508/10$02.00/0

Regulation of Phosphate Accumulation in the Unicellular Cyanobacterium Synechococcus JOHN F. GRILLOt AND JANE GIBSON* Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 Received for publication 17 August 1979

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acidinsoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10%o of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent Km of about 0.5 ,M, but Vm. was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 150C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity. The importance of phosphate supply in regulating the growth of aquatic organisms is widely recognized and has prompted a number of investigations into the uptake and distribution of phosphate in eucaryotic (1, 10, 11, 34, 35) and procaryotic (4, 5, 13, 20, 21, 37) microorganisms. Such studies have emphasized that the phosphate content of whole cells and of individual cell components can be varied over quite wide limits and also that phosphate uptake capacity depends on the previous history of the cells (4, 13, 38, 42). This is also true of polyphosphate, which can be accumulated to the extent of 50% or more of the total cell phosphorus if celLs are provided with energy and phosphate in excess but are restricted by some other nutrient, such as nitrogen or sulfate (16, 18), or if the cells are supplied with phosphate after a period of deprivation (1, 13, 40). Cyanobacteria are frequently prominent members of undesirable blooms in natural waters, in which phosphate availability is a comt Present addres: Department of Microbiology, University of Texas, Austin, TX 79712.

mon limiting factor, and their phosphate relationships have been studied by a number of

workers. Although continuous culture methods were employed in some of these investigations (5, 21), the physiological state of the cells was less well defined in others (4, 20, 41), so that it is not always clear whether the observations made relate specifically to phosphate starvation or to nutrient restriction in general. The work described here made use of balanced exponential cultures growing without nutrient restriction and also of nitrate- and phosphate-limited continuous cultures for investigations into the regulation both of phosphate uptake and of its intracellular fate in the unicellular cyanobacterium Synechococcus. The main findings are that enhanced uptake and potential for polyphosphate formation are similarly regulated, but are distinct processes. MATERIALS AND METHODS The strain of Synechococcus and the general culture conditions and media used have been described previously (23). Water-jacketed vessels (200- or 400-ml working volume), fitted with inlet ports for medium

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PHOSPHATE UPTAKE IN SYNECHOCOCCUS

and for the C02-air mixture and with a single outlet port for gases and culture (25), were used for continuous culture, and all flexible connections were made with surgical-grade silicone tubing. The nitrate-limited medium contained 0.25 mM KNO3 and 1 mM potassium phosphate, pH 8; 15 IM potassium phosphate and 2.5 mM KNO3 were used for phosphate-limited cultures. Both media also contained 15 mM triethanolamine brought to pH 8 with H2S04. Each vessel was illuminated by two 100-W incandescent bulbs at a distance of approximately 7 cm. Flow rates were maintained by Harvard peristaltic or syringe pumps. Approach to a steady state was followed by monitoring optical density at 750 nm in a Zeiss PMQ II spectrophotometer and by protein determination (29); a steady state was assumed when these parameters had remained constant for five volume changes in the culture vessel. A similar rule of thumb was followed after the dilution rate was changed. At steady state, the specific growth rate (In 2/doubling time) equals the dilution rate (22). Photosynthetic pigments were estimated as described previously (23). Phosphate distribution and estimation. Pi was estimated as described by Dryer et al. (7). Total phosphate was measured by using the same method after digestion of cell material by autoclaving in 10% (wt/ vol) K2S208 in tightly closed screw-capped tubes for 45 min at 121°C (4). Radioactively labeled cells were fractionated as described by Roberts et al. (36), except that CHC13-CH30H (3:1, vol/vol) was used for lipid extraction and RNA was solubilized by incubation in 0.3 N KOH for 16 h at 370C before DNA was hydrolyzed in hot 5% trichloroacetic acid. Polyphosphate and nucleic acid-derived phosphate in these fractions were separated by charcoal adsorption of nucleotides. Total phosphate in each fraction was obtained by K2S208 digestion of an appropriately sized portion. As used here, the term polyphosphate refers only to cold acid-insoluble polymer. 32P uptake. In long-term uptake experiments, washed cell suspensions from batch or continuous cultures were transferred to warmed medium preequilibrated with 1% C02-air and containing 0.2 to 0.15 mM phosphate (0.2 to 0.7 MCi/,umol). Cultures were diluted with temperature- and gas-equilibrated medium at intervals so that cell densities were kept between the limits of 30 and 100 ,ug of protein per ml throughout the growing periods. Short-term uptake was measured at 300C or as given in individual experiments. Cells were washed and diluted in 10 mM triethanolamine sulfate, pH 8.0. Neither rates nor affinities were affected by the addition of 10 mM KCl or 5 mM MgCl2 or by changing the pH of the assay mixture to 7.5. Radioactive phosphate (1 to 50 ,Ci/,umol) was added to initiate uptake, and five or six 0.5-ml samples were taken in each measurement at intervals of 10 s to 2 min, depending on the experiment. Each sample was filtered rapidly through a 0.45-pin pore size membrane filter which had been boiled previously in 0.5 M lithium chloride-1 mM phosphate, pH 9, washed twice with 0.5-ml portions of the same solution, and dried at 70°C. For experiments in which the proportion of total counts taken up recoverable as Pi were to be measured, alternate filtered samples were transferred as rapidly as possible to weighing dishes containing 2-ml portions of 5%

509

trichloroacetic acid or 0.5 N perchloric acid at 00C. After 30 min, the samples were centrifuged, and portions of the supernatant were taken for total counts and for determination of Pi by precipitation of the phosphomolybdate complex with triethylamine (43, 46) or extraction into isobutanol-benzene (1:1, vol/ vol) (3). The remaining samples, filtered, washed, and dried as usual, served as controls for checking total uptake in these experiments. Uptake was linear with time in each experimental series. Radioactivity was usually measured either on dried membrane filters or on samples of radioactive solution dried on 2.5-cm disks of Whatman no. 3 filter paper in 0.4% (wt/vol) 2,5-bis(5'-t-butyl 2-benzoxazolyl)thiophene in toluene. Counts in butanol-benzene extracts were measured in 0.2 N NH40H by Cherenkov radiation. Specific activities were determined from samples extracted and counted in the same way. Cell breakage and enzyme assays. Suspensions containing 5 to 20 mg of cell protein per ml in G buffer [0.02 M Tris-sulfate, pH 8, 0.2 M (NH4)2SO4, 1 mM EDTA, 1 mM ,B-mercaptoethanol; modified from Muhlradt (32)] were passed twice through a chilled French pressure cell at 12 to 14,000 lb/in.2 All subsequent steps were carried out at 0 to 40C. Unbroken cells and large debris were removed by centrifugation at 11,000 x g for 20 min. The supernatant was centrifuged again for 90 min at 96,000 x g, and the precipitate of green membranes was discarded. Solid (NH4)2SO4 (2.25 g/10 ml) was dissolved in the supernatant, and the whole solution was allowed to stand in ice for 30 min. The heavy precipitate, which contained more than 90% of the polyphosphate synthetase and polyphosphatase activities of the extract, was collected by centrifugation at 11,500 x g for 30 min. It was dissolved in 1 to 3 ml of G buffer and assayed at once. In one preparation 1 mM phenylmethyl sulfonyl fluoride was included in the buffer in which the cells were broken to inhibit protease activity which might be responsible for releasing membrane-bound proteins into the soluble portion. This did not affect the distribution of either enzyme activity after high-speed centrifugation. Although polyphosphatase activity was unchanged after 3 weeks of storage in ice, about 30% of the polyphosphate synthetase activity was lost in 1 day, either in ice or frozen at -20°C. Inactivation was faster if (NH4)2SO4 or fB-mercaptoethanol was omitted from the suspending buffer. Neither enzyme activity decreased by more than 15% if whole cells were kept frozen at -60C for 6 weeks. Polyphosphate synthetase. Polyphosphate synthetase was assayed by determining the rate of formation of acid-insoluble radioactive material from [y32P]ATP (26, 32), using 0.2 to 2 mg of extract protein in a reaction volume of 0.5 ml. Preliminary experiments established that Mg2' was required for the reaction and that 5 mM ATP, with a ratio of Mg2e to ATP of at least 1:1, was optimal. Increase in acidinsoluble radioactivity was linearly related to time and to enzyme concentration under these conditions. Addition of polyphosphate as a primer for the reaction was not required and did not alter the rate of the reaction. ADP (2 mM) added to the reaction mixture reduced the counts in acid-precipitable material by 85%, suggesting that the reaction was reversible.

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The reaction product was characterized by heating for 20 min at 100°C in 1 N HCI (14), after protein and nucleic acid were removed by adsorption to charcoal in the presence of 0.5 m NaCl (32); 87% of the total counts in the original preparation were recovered as Pi after this treatment. The deproteinized product was also used for estimating metachromasy in toluidine blue 0 (45) and gave the same absorbance shifts at 530 and 630 nm expressed in terms of phosphate content as a sample of polyphosphate isolated chemically from Synechococcus, as described below. Polyphosphatase. Polyphosphatase was assayed by following release of Pi from chemically isolated polyphosphate (12) in 1-mil reaction mixtures containing 0.3 to 1.4 mg of extract protein and polyphosphate equivalent to 1.5 to 2 pmol of acid-releasable phosphate. Preliminary experiments showed that Mg2" was required in this reaction. Preparation of polyphosphate. Phosphate-free medium (3 liters) was inoculated with cells from an exponential culture to a protein concentration of 175 ,ug/ml. After 72 h of starvation in light and with airCO2 gassing, Pi to a final concentration of 5 mM was added, and the accumulation of polyphosphate was allowed to proceed for 5 h. The whole culture was then chilled, and the cells were collected by centrifugation and stored at -60°C until used. Radioactively labeled polyphosphate was prepared from a 200-ml culture starved as described above and then supplied with 0.135 mM phosphate (12 utCi/jLmol) and incubated for an additional 5.5 h. Only 3% of the counts added were left in the medium at this time. Polyphosphate was extracted from hypochlorite digests of cells as described by Harold (14). The final preparation was faintly opalescent and contained no detectable Pi before acid hydrolysis. The material was completely excluded from Sephadex G-50 and 85% was excluded from Sephadex G-75, suggesting that the average molecular weight was more than 50,000.

Internal water space. Internal water space was measured as previously descibed (23). Radioactive materials. 32P; carrier-free in 0.1 N HCI was obtained from New England Nuclear Corp. or from International Chemical and Nuclear Co. The [y-32P]ATP used in the enzyme assays was a gift from Ray Wu, Cornell University.

RESULTTS Cell composition: steady states. When the composition of cells grown either without restriction or with limiting nitrate is compared with the composition of phosphate-restricted cells (Table 1), it is apparent that most alterations in phosphate content can be attributed specifically to phosphate restriction rather than to changed growth rate. The RNA content is exceptional in varying directly with growth rate in both types of restriction, even though the absolute level was consistently lower in the phosphate-limited cultures. The Pi pool (Table 2) was reduced almost to one-tenth of that found in unrestricted cells at the lowest phosphatelimited specific growth rate investigated. It was, however, still more than 1 mM, although the culture outflow phosphate was below 5 ,uM, the limit of detection with the method used. Cultures whose growth rate was restricted by the availability of either phosphate or nitrate were yellow-green and had greatly reduced phycocyanin contents, as previously described (2, 24); phycocyanin content varied directly with specific growth rate over the range of 0.025 h-' (50 ,ug of phycocyanin per mg of total protein) to 0.163 h-' (200 ug of phycocyanin per mg of

TABLE 1. Phosphate content of Synechococcus cell fractions Growth condition

Phosphate content (umol/mg) of the following fractions:" Specific growth Chloroform Cold acid sol- Chloroformrate RNA DNA methanol hae (h') ubleb phated (h-')

~solublec

Balanced batch culture

0.14

0.136 (17.7)e 0.038 (4.9)

Phosphate-limited continuous culture

0.115 0.07 0.06 0.05 0.025

0.094 (16.8) 0.075 (16) 0.065 (18) 0.063 (16.4) 0.054 (21)

0.031 (5.5) 0.028 (6.0) 0.024 (6.6) 0.016 (4.2) 0.019 (7.4)

0.051 (6.6)

0.42 (54.4)

0.081 (11)

0.055 (9.8) 0.05 (10.7) 0.05 (13.9) 0.061 (15.9) 0.048 (18.8)

0.317 (56.7) 0.241 (51.5) 0.2 (55.4) 0.181 (47.1) 0.096 (37.5)

0.05 (8.9) 0.058 (12.4) 0.043 (11.9) 0.04 (10.4) 0.037 (14.5)

0.087 (11) 0.05 (6.3) 0.051 (6.5) 0.515 (63.3) 0.007 (0.9) 0.016 (2.3) 0.122 (17.3) 0.042 (6.2) 0.048 (6.8) 0.396 (56) 0.361 (49.6) 0.063 (8.6) 0.044 (5.8) 0.05 (6.9) 0.16 (22) 0.158 (20) 0.045 (5.8) 0.052 (6.7) 0.231 (30) 0.173 (22) aMicromoles of phosphate per milligram of total cell protein. b Cold 5% trichloroacetic acid. c Chloroform-methanol, 3:1. d Cold acid (5% trichloroactic acid)-precipitable polyphosphate. 'Numbers in parentheses are phosphate content expressed as percentage of the chemically determined total in the cells.

Nitrate-limited continuous culture

0.163 0.115 0.064 0.032

VOL. 140, 1979

PHOSPHATE UPTAKE IN SYNECHOCOCCUS

TABLE 2. Cold acid-soluble pools in balanced exponential and phosphate-lunited continuous cultures Acid-soluble pool' Spe-

Growth c tion

cific growth rate

(h-1)

Balanced exponential culture Phosphatelimited continuous cul-

Other phosphate Concn % of Concn % of (mM) total (mM) total

0.14b

11.4

42

15.4

58

0.115C

4.2 2.4 1.4

28 25 21

11.1 7.1 5.3

72 75 79

0.05d

0.025' tures a The values given are averages of three or more determinations except for the balanced exponential which are averaged duplicate results. results, b Internal volume, 5.1 of protein (23). " Internal volume, 4.9 pl/mg of protein. d Internal volume, 4.6 pl/mg of protein. Internal volume, 4.2 pl/mg of protein.

id/mg

511

Figures 3A and B show that addition of phosphate to starved cultures did not lead to the direct restoration of the normal phosphorus distribution, but rather involved uptake to at least twice the ratios of phosphate to protein found in exponential cells. Between 3 and 7 h after the addition, more than 60% of the newly acquired phosphate was found as polyphosphate, and rapid incorporation into RNA was also seen (Fig. 3B). Net protein synthesis resumed at an exponential rate between 8 and 10 h after phosphate was added, and at the same time the proportion of counts in polyphosphate began to decline toward normal levels, which were reached after about 35 to 40 h. Polyphosphate breakdown occurred despite the continued availability and uptake of Pi from the medium (Fig. 3A). Cells from phosphate-limited continuous cultures also showed excess phosphate uptake when transferred to phosphate-containing medium, to an extent that depended on the specific growth rate and initial ratio of phosphate to protein (Table 3). Again, polyphosphate accounted for

protein). Cells growing at very low specific growth rates contained up to 15 times more carbohydrate than cells from unrestricted cul- a tures, based on cell protein (data not shown). Despite such changes, growth could be main10 tained indefinitely. S S Cell composition: transients. If balanced ~0 exponential cultures which had been uniformly Ilabeled by growth for five doublings in excess Pi containing 32p043- were transferred to medium containing the same concentration of unlabeled 1-!' wE Pi, the counts initially in the acid-soluble pools x were chased within one doubling time (5 h), 0. lag mainly into RNA. No loss of counts from RNA, (4 DNA, or the polyphosphate fraction was ob- Cf, served over the subsequent 20 h of continued exponential growth (Fig. 1). No labeled phosphate was found in the medium in these experiments. When prelabeled cells were transferred to medium without added phosphate, there was a TIME hours progressive and approximately proportionate FIG. 1. Turnover of phosphorus-containing fracloss of phosphate from all cell fractions including tions in Synechococcus maintained in a quasi-steady polyphosphate (Fig. 2). Again, no label was de- state by intermittent dilution of the culture during tected extracellularly. Net protein synthesis growth. Cells were labeled by growth for 24 h in stopped after about 30 to 36 h, by which time medium containing 0.15 mM K2HP04 (1.5 liCi/Mmol) RNA counts had been reduced to one-quarter of to a finalprotein concentration of 83 pg/ml. The ceUs and transferred to unlabeled medium the starting value and the culture color had were washed0.163 mM K2HP04; the protein content containing changed to yellow-green. As had been found was 20 ug/ml. During experiment, the protein previously (24), the culture remained viable, and content of the culture wastheheld between 12 and 41 pg/ growth resumed on readdition of phosphate ml by dilution with medium of the same composition. after a lag, the length of which depended on the Symbols: 0, cold acid soluble; x, RNA; A, DNA; U, duration of the starvation period. CHCl3-CH30H soluble; *, polyphosphate; 0:, protein. l

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J. BACTERIOL.

GRILLO AND GIBSON

T~~~~~~~~~~~~ /

10~

-

one-tenth this concentration. Arsenate at 100fold excess over phosphate was without effect (Table 5). All of the radioactivity associated with cells after uptake periods of 1 to 2 min at 150C was acid soluble. Pi accounted for 93.3 ± 7.3% of the total counts in a total of 13 samples in several experiments taken between 11 and 50 s after uptake was started. At 20 and 260C, 85 to 90% of the extracted counts were Pi in samples taken within 30 s, but movement both into other components of the acid-soluble pool and into acidinsoluble components was apparent between 1 z

-7

0-0~

Li a.

z

w LU

TIME (hours)

FIG. 2. Changes in phosphorus content of cell fractions during phosphate starvation of Synechococcus. Cells were labeled by growth for 24 h in medium containing 0.163 mM K2HIPO4 (2.6 ,tCi4&lmol) to a final protein concentration of 83 p/mi. The cells were washed and transferred to phosphate-free medium; theprotein concentration was 16pg/ml. During the experiment, the protein content of the culture was held between 15 and 65 pg/mi by dilution with phosphate-free medium. Symbols: 0, cold acid soluble (x102); x, RNA (x103); A\, DNA (x102);U, CHCI3CH3OH soluble (x102); 0, polyphosphate (x102); 0, protein.

I

,P

Ec

a

aww

50 to 60% of the phosphate accumulated after 5 0 to 6 h (data not shown). w Phosphate uptake kinetics. The initial rate a.ul of phosphate uptake also depended on the -J growth conditions. Cells from all types of cul0 tures showed Michaelis-Menten uptake kinetics saturable at low concentrations. Kinetic constants obtained from double-reciprocal plots of 20 30 10 0 experimnental results when cells grown under TIME hours different conditions were used are summarized FIG. 3. (A) Totalphosphate uptake and net protein in Table 4. Vms1,, was substantially greater in synthesis after ) mMPi was added to 24-h phosphatephosphate-restricted cells, but the apparent Km starved cells. (B) Distribution of new phosphate into was not significantly affected by growth condi- ceU fractions. An exponential culture was harvested tions. The increased maximnum uptake rate was and used to inoculate phosphate-free medium; the clearly the result of phosphate limitation, since protein concentration was 14 pg/ml. The cells were the apparent V,l,, of nitrate-limited cells of com- starved for 24 h (final protein concentration, 46 pg/ parably low specific growth rates was close to ml), then harvested and used to inoculate labeled that of unrestricted, rapidly growing cells. Phos- medium containing 0.17 mM K2HPO4 (0.9 tCi/wmol) phate uptake rates were reduced by 40% if cells to 23 pg ofprotein per ml. The protein content of the was maintained between 23 and 88 pg/ml by were not illuminated, but virtually abolished if culture when necessary with medium of the same cells were kept both dark and anaerombic. The dilution composition and specific activity. (A) Symbols: 0, cell uncoupler carbonyl cyanide-p-trifluoromethoxy protein; o, 32P taken up per milliliter of culture; 0, phenyihydrazone inhibited uptake at concentra- 2p taken up per microgram ofprotein. (B) Symbols: tions of more than 20 ,uM, and the fluorine 0, cold acid soluble; 0, RNA; A, DNA; , CH3Clanalog FCCP was effective at approximately CH30H soluble; 0, polyphosphate.

PHOSPHATE UPTAKE IN SYNECHOCOCCUS

VOL. 140, 1979

TABLE 3. Relation between phosphate-restricted specific growth rate, initial ratio ofphosphate to protein, and extent ofphosphate accumulationa Specific growth rate

(h'-)

Ratio of phosphate to protein (anol/mg)

Initial 0.75 0.56 0.47 0.38 0.26

0.14 b 0.115c 0.07c 0.05c 0.025c

Maximal

0.75 0.94 1.15 1.56 1.86 a Samples from continuous cultures were transferred to 100 ml of medium containing 0.125 to 0.194 mM phosphate (0.45 to 1.8 uCi/ftmol) as described in the text to a final protein concentration of 20 ltg/ml. Samples were taken over a 24-h period, initially at 30min intervals, filtered through membrane filters, washed, and counted. b Balanced exponential culture (control). Phosphate-limited continuous cultures.

TABLE 4.

Kinetics ofphosphate uptake in Synechococcusa

513

phosphate was found as polyphosphate, the amount of this material in the cells presumably reflecting the relative activities of polyphosphate-forming and -degrading enzyme systems. Polyphosphate synthesis could indeed be measured in extracts prepared from all cells, but the rates of formation were substantially greater in preparations from phosphate-restricted or -starved cultures (Table 6). The changed activity could be attributed specifically to phosphate limitation, since extracts from nitrate-limited continuous culture had specific activities close to those from unrestricted cells. In contrast, although the specific activity of polyphosphatase in extracts from cells grown in continuous culture was about twice that of batch cultures, this was true for both phosphate- and nitratelimited conditions and was also independent of growth rate (Table 6). Figure 4 shows the changes in specific activity of polyphosphate synthetase in extracts prepared at intervals during phosphate starvation and recovery after addition of 1 mM Pi. A pro-

V..c

Growth condition cniin

Balanced batch Nitrate limited Phosphate limited

Specific Concn of growth rate (h'1) Pi pool Km

(MMI~)b

0.14 0.064

11.4 8.6

(YM)¢

0.2 1.0

(nmol/ min per mg of protein)

TABLE 5. Effect ofpossible inhibitors on phosphate uptake by phosphate-limited chemostat-grown ceilsa Uptake rate

1.3 2

2.4 0.3 53 1.4 0.25 48 a Phosphate uptake was measured in light at as described in the text. Celis from balanced batch and nitrate-limited continuous cultures were collected by filtration and washed in 10 mM triethanolamine sulfate, pH 8, before being suspended in the same solution to between 6 and 20 yig of protein per ml. Phosphatelimited cultures were diluted directly into buffer to a protein concentration of 1.3 to 1.7 ug/ml. Uptake was initiated by adding phosphate having a specific activity of 0.05 to 0.25 ,LCi/nmol. b Determined on separate samples from the same

0.05 0.025

250C

cultures. 'From Lineweaver-Burk plots.

and 2 min. These results suggest strongly that Pi is brought into the cells as such and moves into other phosphate-containing components after mixing with the intracellular Pi pool. Polyphosphate formation. Cells from balanced exponential cultures and from restricted cultures all contained some polyphosphate, although this amounted to a substantial part of the total cell phosphate only in the nitrate-limited continuous culture with the lowest specific growth rate. However, even in very slow phosphate-limited growth, about 10% of the cell

Condition

Addition

(inole/r minpe

mg of protein)

Light control Dark, aerobic Dark, anaerobic

% f control value

100 56 3 Arsenate (100 M) 95 CCCP (25 uM)b 70 CCCP (50 uM) 39 CCCP (100MAM) 2 Phosphate concentration, 1puM; specific growth rate, 0.025 h-'. Inhibitors were added 5 min before uptake was started. b CCCP, Carbonyl cyanide-m-chlorophenyl hydrazone. 32.8 18.3 1.0 31.2 28.8 12.8 0.7

TABLE 6. Specific activities ofpolyphosphate synthetase and polyphosphatase in Synechococcus grown under different conditions Sp act of polyphos growth rate Pphate rth' (hW1) synthe-

Specific

Growth condition

Sp act of

poly-

phosphatasea

tasea

Balanced exponential culture Continuous culture, phosphate limited

0.14 0.115 0.05 0.025 0.064

1.4

8.7

15 19.4 24.9 22.3 34.2 20.5 2.2 23.9 Nitrate limited a Enzyme activities are given as nanomoles of product formed per minute per milliam of protein at

300C.

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GRILLO AND GIBSON

J. BACTERIOL.

the cells. Although polyphosphatase showed saturation kinetics, maximum activity required high concentrations of substrate, and an apparent Km of 12 mM (expressed as acid-releasable Pi) was obtained from double-reciprocal plots. Even at polymer concentrations which supported only about one-third of the maximum rate, the time course of Pi release was linear until 70 to 80% of the total polyphosphate had been hydrolyzed. This suggests that the substrate concentration was little changed during the course of the hydrolysis, as would be the case if the enzyme were an exophosphatase able TIME hours to attack the polymer only at chain ends. This FIG. 4. Changes in cell phosphate and specific ac- suggestion was supported by an experiment in tivities of polyphosphate synthetase and polyphos- which samples from an enzymatic digestion of phatase in a culture starved for 50 h and after sub- 32P-labeled polyphosphate were removed and sequent readdition of phosphate at point shown by chromatographed on polyethyleneimine at inarrow. Three Roux bottles, each containing 800 ml of phosphate-free medium, were inoculated to 50 pg/ml tervals until digestion was complete. Only radiowith washed cells from an exponential culture grow- active material which stayed at the origin and ing in a medium containing 0.25 mM K2HP04, incu- an increasing amount of Pi could be detected bated in light at 30°C, and bubbled with air-CO2. At (data not shown). the intervals shown equal volumes from each bottle (40 to 100 mg of total protein) were filtered through 0.45-p,1m polypropylene filters, washed two times with 10 ml of deionized water, and stored at -60°C until used. Phosphate-free, warmed medium was added to the cultures to restore the original volume. Samples for protein determination and for total cellphosphate contained 0.04 to 0.1 and 0.25 to 0.8 mg of cel protein, respectively. After the stored cells were removed from the filters by agitation in 9 ml of G buffer, extracts were prepared and assayed as described in the text.

gressive increase in the activity of the enzyme to about 13 times that found at the start of the experiment was observed, so that its activity rose in parallel with the capacity of the cells to accumulate polyphosphate. After the readdition of phosphate, specific activity declined until the original value was reached after about 40 h. The decrease in specific activity was most rapid in the first 8 h after phosphate addition and preceded the resumption of net protein synthesis, which was not observed until between 8 and 16 h. Again, the polyphosphatase activity of extracts did not change significantly during the whole course of the experiment. The almost constant specific activity of polyphosphatase in all extracts appeared to be inconsistent with the markedly different stability of intracellular polyphosphate observed in the experiments described above. Possible ways in which existing potential enzyme activity could be regulated were therefore investigated. Pi release from radioactively labeled polyphosphate by dialyzed extracts was inhibited by at most 15% by the addition of up to 20 mM Pi to the assay mixture, showing the polyphosphatase was not regulated by variation in the Pi pool within

DISCUSSION Many microorganisms accumulate excess phosphate, generally in the form of polyphosphates, under special conditions (16). The major aims in the present investigation were to examine phosphate distribution in the phototrophic procaryote Synechococcus in balanced growth under a range of different conditions and to attempt to account for the findings in terms of three contributing cellular activities: phosphate uptake across the cell membrane, polyphosphate synthetase, and polyphosphatase. Specific effects of phosphate restriction on the composition of cells grown in continuous culture at similar growth rates become obvious from comparisons between phosphate- and nitratelimited cultures. Growth rate restriction affects the RNA content with either limitation, but is more obvious when phosphate is limiting. This suggests that ribosome content is less rigorously correlated with growth rate in Synechococcus than, for example, in Escherichia coli and Salmonella typhimurium (8, 30). A significant part of the total cell phosphate was found to be sequestered as polyphosphate even at the lowest specific growth rate studied. Cells from phosphate-restricted growth took up phosphate to well above the levels found in unrestricted cells when Pi was added in excess, and polyphosphate accounted for about 60% of the total in the cells at the time when this total was at its peak. Measurements of individual activities showed that phosphate limitation specifically increased (by factors of 30 to 50 times) both the maximum rate at which phosphate was transported into the cells and the specific activity of polyphos-

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phate synthetase in extracts in cells from steady states and in cells from unrestricted growth transferred to phosphate-free medium for periods of about 40 h. No change in polyphosphatase activity occurred. An expected consequence under steady-state conditions would be that the efficiency with which cells could acquire phosphate from the medium would be raised, but that at the same time polyphosphate synthesis would compete more effectively with other, functionally more important, phosphate-utilizing reactions in such cells. The increased activities would also account for the rapid phosphate accumulation and polyphosphate formation seen when medium phosphate concentration was raised. The high transport and polyphosphate synthetase activities characteristic of phosphate restriction were not maintained once Pi was resupplied, and both returned to values characteristic of unrestricted or nitrate-limited balanced cultures within about 6 to 8 h, that is, before significant net protein synthesis had occurred. This suggests either that there is specific inactivation of these cell components or that they have a quite rapid turnover under all conditions. In either case, these activities, like alkaline phosphatase (24) and glucose 6-phosphate dehydrogenase (33) activities, are clearly regulated in response to specific growth conditions. Alkaline phosphatase was measured with other activities in this study (data not shown). With the exception that the specific enzyme activity appeared to decrease only after the resumption of net protein synthesis in refeeding experiments and could thus be accounted for primarily in terms of dilution of existing stable enzyme, the changes in alkaline phosphatase were parallel to those in transport and in polyphosphate synthetase. These activities thus appear to be coordinately regulated in wild-type cells of Synechococcus, as they are in Aerobacter aerogenes where, however, polyphosphatase is also included (15, 16). The potential for polyphosphate formation is not an essential characteristic (19), and mutants of Synechococcus in which this is absent have been described recently (44). A study of the other phosphaterelated activities described in this paper in such strains would be of obvious help in understanding the genetic relationships. The precise nature of the intracellular signal which regulates the expression of these activities is also as yet unknown. Although the size of the intracellular Pi pool can indeed be varied over about a 10-fold range, it seems at least as probable that a more sensitive indicator metabolite, as yet unknown, may be involved, as Willsky et al. (47) have suggested for E. coli.

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The almost unvarying activity of polyphosphatase found in these experiments differs from findings with other systems (15, 16, 27, 28). It was also unexpected since polyphosphate does not turn over during unrestricted growth and is not selectively mobilized during phosphate starvation. On the other hand, it is rapidly eliminated after excess formation has occurred after phosphate restriction; this breakdown becomes evident as soon as uptake and synthesis activities return to normal levels. In view of its kinetics, it is possible that substrate concentration may normally restrict the activity of this enzyme, which appears to be an exophosphatase, as it is in Corynebacterium xerosis (31). However, other explanations, such as a different location of the small amount of polyphosphate normally present and excess accumulation, can also be suggested. Phosphate was transported as Pi against high concentration gradients by Synechococcus in an energy-dependent manner no matter what growth conditions were used. High affinity and energy dependence are in fact generally observed in a number of both procaryotic and eucaryotic microorganisms (1, 4, 6, 9, 17, 20, 21, 34, 38, 41), although the reported values for kinetic constants vary somewhat, and complex kinetics have.been observed in multicompartmented eucaryotic cells (35, 39). The apparent Km for uptake measured here did not change, although Vmax could be increased by a factor of at least 30-fold by a history of phosphate restriction. This suggests, although it does not prove, that differential expression of a single system is involved. In E. coli, two phosphate transport systems, one inducible and one constitutive, are found (38, 47, 46); these differ also in kinetic constants and in arsenate sensitivity. Phosphate uptake in Synechococcus was virtually unaffected by a 100-fold excess of arsenate, and the cells were found to grow as well in 20 ,uM phosphate-50 mM arsenate as in phosphate alone, so that attempts to obtain mutants by using arsenate resistance were not successful. ACKNOWLEDGMENTS This work was supported by grants BMS 7301836 and 7707443 from the National Science Foundation and by funds provided by the New York State Agricultural Experiment Station. LITERATURE CITED 1. Aitchison, P. A., and V. S. Butt. 1973. The relation

between the synthesis of inorganic polyphosphate and phosphate uptake by Chlorella vulgaris. J. Exp. Bot. 24:497-510. 2. Allen, M. M., and A. J. Smith. 1969. Nitrogen chlorosis in blue-green algae. Arch. Mikrobiol. 69:114-120. 3. Avron, M. 1960. Photophosphorylation by swiss chard chloroplasts. Biochim. Biophys. Acta 40:257-272.

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4. Batterton, J. C., and C. Van Baalen. 1968. Phosphorus deficiency and phosphate uptake in the blue-green alga Anacystis nidulans. Can. J. Microbiol. 14:341-348. 5. Bone, D. H. 1971. Relationship betwen phosphates and alkaline phosphatases of Anabaena flos-aquae in continuous culture. Arch. Mikrobiol. 80:147-153. 6. Bornefeld, T., and W. Simonis. 1974. Correlations between phosphate uptake, photophosphorylation and metabolism in Anacystis nidulans as affected by carbonylcyanide m-chlorophenylhydrazone and chloramphenicol, p. 1557-1565. In M. Avron (ed.), Proceeding of the Third International Congress on Photosynthesis, vol. 2. Elsevier Scientific Publishing Co., Amsterdam. 7. Dryer, R. L., A. R. Tammes, and J. I. Routh. 1957. The determination of phosphorus and phosphate with Nphenyl-p-phenylenediamine. J. Biol. Chem. 225:177183. 8. Edlin, G., and P. Broda. 1968. Physiology and genetics of the "ribonucleic acid control" locus in Escherichia coli. Bacteriol. Rev. 32:206-226. 9. Falkner, G., K Werdan, F. Horner, and H. W. Heldt. 1974. Energieabhangige Phosphataufnahme der blaualge Anacystis nidulans. Ber. Dtsch. Bot. Ges. 87: 263-266. 10. Fitzgerald, G. P. 1969. Field and laboratory evaluations of bioassays for nitrogen and phosphorus with algae and aquatic weeds. Limnol. Oceanogr. 14:206-212. 11. Fuhs, G. W., S. D. Demmerle, E. Canelli, and M. Chen. 1972. Characterization of phosphorus-Uiited plankton algae (with reflections on the limiting nutrient concept), p. 113-133. In G. E. Likens (ed.), Nutrients and eutrophication. The limiting nutrient controversy. American Society of Limnology and Oceanography Special Symposia, vol. 1. Allen Press, Lawrence, Kan. 12. Gezelius, K. 1974. Inorganic polyphosphates and enzymes of polyphosphate metabolism in the cellular slime mold Dictyostelium discoideum. Arch. Microbiol. 98:311329. 13. Harold, F. M. 1963. Accumulation of inorganic polyphosphate in Aerobacter aerogenes. I. Relationship to growth and nucleic acid synthesis. J. Bacteriol. 86:216221. 14. Harold, F. M. 1963. Inorganic polyphosphate of high molceular weight from Aerobacter aerogenes. J. Bacteriol. 86:885-886. 15. Harold, F. M. 1964. Enzymic and genetic control of polyphosphate accumulation in Aerobacter aerogenes. J. Gen. Microbio. 35:81-90. 16. Harold, F. M. 1966. Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol. Rev. 30: 772-794. 17. Harold, F. M., and E. Spitz. 1975. Accumulation of arsenate, phosphate, and aspartate by Streptococcus faecalis. J. Bacteriol. 122:266-277. 18. Harold, F. M., and S. Sylvan. 1963. Accumulation of inorganic polyphosphate in Aerobacter aerogenes. II. Environmental control and the role of sulfur compounds. J. Bacteriol. 86:222-231. 19. Harold, R. L., and F. M. Harold. 1963. Mutants of Aerobacter aerogenes blocked in the accumulation of inorganic polyphosphate. J. Gen. Microbiol. 31:241246. 20. Healey, F. P. 1973. Characteristics of phosphorus deficiency in Anabeana. J. Phycol. 9:383-394. 21. Healey, F. P., and L. L Hendel. 1975. Effects of phosphorus deficiency in two algae growing in chemostats. J. Phycol. 11:303-309. 22. Herbert, D., R. Elsworth, and R. C. Telling. 1956. The continuous culture of bacteria; a theoretical and experimental study. J. Gen. Microbiol. 14: 601-622. 23. Ihlenfeldt, M. J. A., and J. Gibson. 1975. CO2 fixation and its regulation in Anacystis nidulans (Synechococcu8). Arch. Microbiol. 102:13-21.

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J. Cedergren. 1978. Polyphosphage-deficient mutants of Anacystu nidulans. Can. J. Microbiol. 24:112-116. 45. Wiame, J. M. 1948. The occurrence and physiological behavior of two metaphosphate fractions in yeast. J. Biol. Chem. 178:919-929. 46. Wilkin8, A. S. 1972. Physiological factors in the regulation of alkaline phosphatase synthesis in Escherichia

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coli. J. Bacteriol. 110:616-623. 47. Willsky, G. R., R. L, Bennett, and M. H. Malamy. 1973. Inorganic phosphate transport in Escherichia coli: involvement of two genes which play a role in alkaline phosphatase regulation. J. Bacteriol. 113:529539.