phosphate uptake in the cyanobacterium synechococcus r-2 pcc

Plant Cell Physiol. 38(11): 1232-1241 (1997)JSPP © 1997

Phosphate Uptake in the Cyanobacterium Synechococcus R-2 PCC 7942

Raymond J. Ritchie, Donelle A. Trautman and A.W.D. LarkumBiology A-12, School of Biological Sciences, The University of Sydney, NSW 2006, Australia

Phosphate uptake rates in Synechococcus R-2 in BG-11 media (a nitrate-based medium, not phosphate limited)were measured using cells grown semi-continuously and incontinuous culture. Net uptake of phosphate is propor-tional to external concentration. Growing cells at pH010have a net uptake rate of about 600 pmol m~2s"1 phos-phate, but the isotopk flux for 32P phosphate was about 4nraol m~2s~'. There appears to be a constitutive over-ca-pacity for phosphate uptake. The Km and Vmtx of the satura-ble component were not significantly different at pH0 7.5and 10, hence the transport system probably recognizesboth H2PO4" and HPO2,". The intracellular inorganic phos-phate concentration is about 3 to 10 mol m~3, but there isan intracellular polyphosphate store of about 400 mol m~3.Intracellular inorganic phosphate is 25 to 50 kJ mol'1 fromelectrochemical equilibrium in both the light and darkand at pHo 7.5 and 10. Phosphate uptake is very slow inthe dark (»100 pmol m~2s~1) and is light-activated (pHo

7.5«1.3nmolm"1s~1, pHo 10«600 pmol m"^"1). Up-take has an irreversible requirement for Mg2+ in the medi-um. Uptake in the light is strongly Na+-dependent. Phos-phate uptake was negatively electrogenic (net negativecharge taken up when transporting phosphate) at pHo 7.5,but positively electrogenic at pHo 10. This seems to excludea sodium motive force driven mechanism. An ATP-drivenphosphate uptake mechanism needs to have a stoichio-metry of one phosphate taken up per ATP (1 PO4,0/ATP)to be thermodynamically possible under all the conditionstested in the present study.

Key words: Active transport — Cyanobacteria — Electro-chemical gradient — Membrane potential — Phosphate nu-trition.

The cyanobacterium Synechococcus R-2 has been ex-tensively used as a model organism for many membranetransport studies (Ritchie 1991, 1992a, b, c, 1996, Ritchieand Gibson 1987, Ritchie et al. 1996). Phosphorus is anessential element for all cells but relatively little informa-

Abbreviations: ANOVA, analysis of variance; CAPS, 3-[cy-clohexylamino]-l-propane-sulphonic acid; DMO, 5,5-dimethylox-azoline-2,4-dione; PAR, photosynthetically active radiation; c(subscript), refers to the control-treated cells; e (subscript), refersto the experimental treatment cells; i (subscript), refers to the in-side of the cells; o (subscript), refers to the outside of the cells orbulk electrolyte.

tion is available on the physiological mechanism of uptakein cyanobacteria. Despite the obvious importance of phos-phate nutrition to algal growth under eutrophic conditions,little is known about the physiology of uptake of phos-phate by cyanobacteria (Grillo and Gibson 1979, Falkner etal. 1980, 1989, Rigby et al. 1980, Budd and Kerson 1987,Wagner et al. 1995). Phosphate is often cited as the causeof cyanobacterial blooms but its removal from waste-wateris very expensive, and cause and effect might not be straight-forward.

Many previous studies have worked with cells in experi-mental medium likely to lead to spurious results or resultsthat are not applicable to cyanobacteria growing in nature.For example, Mohleji and Verhoff (1980) used a medium(NAAM) with a very low ionic strength, no buffers and thepH was not specified in their experiments. Grillo and Gib-son (1979) used triethanolamine/H2SO4 (pH 8) buffer.They tested for K+ and Mg2* effects but not for Na+ orCa2+. Tris is known to be toxic to many cyanobacteria, buthas been used in many previous studies. Falkner et al.(1980) used Tris/H2SO4 (pH 7.5) buffer. Ca2+ and Mg*+

effects were investigated but not K+ or Na+. Rigby et al.(1980) and Budd and Kerson (1987) also used Tris/H2SO4

(pH 8). Both studies tested the effects of many differentions upon phosphate uptake including Na+, K+, Ca2+ andMg2"1" but not in combination. The conclusions drawn inthese studies might not have been fully justified. Morerecently, Falkner et al. (1989) and Avendano and Valiente(1994) have used incubation medium containing the sameconstituents as the culture medium plus HEPES/NaOH orKOH buffer and so their results are more likely to havesome relevance.

Although there is little doubt that phosphate is active-ly taken up by cyanobacterial cells (Grillo and Gibson1979, Falkner et al. 1980), a formal thermodynamic analy-sis of phosphate uptake has not been attempted. The aimof this study is to perform a biophysical analysis on themechanism of uptake and the intracellular concentrationsof phosphate under eutrophic conditions with attention topH and Na+, K+ and the divalent cations, Mg2* and Ca2+.Much of the previous work on phosphate nutrition has fo-cused upon phosphate deficient rather than eutrophic con-ditions.

Synechococcus was used in the present study becausemany cyanobacteria have a mucilaginous sheath whichmakes transport studies very difficult or impractical. Un-fortunately, the strains of Anabaena and Microcystis re-sponsible for toxic cyanobacterial blooms are amongst

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Phosphate uptake in a cyanobacterium 1233

those that have thick mucilaginous sheaths making themunsuitable for anything but simple growth studies of nu-trient uptake which are not likely to make great progressin understanding the mechanism of uptake of phosphate incyanobacteria. However, the general findings in the presentstudy should be applicable to understanding the mechan-ism of phosphate uptake in a variety of cyanobacteria in-cluding the more notorious Anabaena and Microcystis.

Materials and Methods

Chemicals and radiochemicals—^Rb"1" was from DUPONTNew England Nuclear, Boston, MA, U.S.A. 32P-H3PO4 wasfrom the Australian Nuclear Science Technology Organization(ANSTO), Lucas Heights, Sydney, Australia. CAPS, DCMU,Fluorinert FC-77, HEPES, methylamine, HC1 and valinomycinwere from Sigma-Aldrich. All reagents used were of analytic stand-ard.

General—Synechococcus R-2 (S. leopoliensis, Anacystisnidulans) (PCC 7942) was grown from axenic stock cultures inBG-11 medium (Allen 1968) modified as described previously (Rit-chie and Gibson 1987, Ritchie et al. 1996) but with the total phos-phate concentration lowered to 50 mmol m~3 and total inorganiccarbon increased to 1 molm"3. The unmodified BG-11 (PO4=175 mmol m~3) precipitates much of its phosphate upon autoclav-ing. Phosphate-free BG-11 contained much less than 0.1 mmolm~3 phosphate. The cells were grown on air, either semi-continu-ously (~ 1/4 dilution each day) or in a continuous culture device,in continuous light of about 150^mol (quanta) m~2 s~l PAR, us-ing cool white fluorescent lights, at 30°C. Similar light conditionswere used in experiments but the temperature was 25°C. The pH0

of a growing culture was about 10 to 10.5.All glass and plastic laboratory-ware were HC1 acid-washed

before use. The cells were usually harvested by centrifugation(2,500 x j ) in a preparative centrifuge and washed three times inthe experimental medium. All experiments were run in media asclose as possible in ionic composition to the BG-11 medium inwhich they had been grown. A routine pre-incubation of 30 minwas run in the light or dark as appropriate, before using cells inthe experiments.

Intracellular volumes, cellular surface areas and numbers ofcells were calculated using stereological data from Ritchie and Gib-son (1987) and Ritchie (1991) using an empirical relationship be-tween light scattering (A75onm), measured using a Varian® Cary13E UV-Visible spectrophotometer.

Buffer solutions—The ionic composition of BG-11 mediumwas changed as little as possible: buffers were adjusted to the ap-propriate pH0 using NaOH or Ca(OH)2 for increasing alkalinity.Ca(OH)2 contains some CaCO3: this needs to be removed by filtra-tion or sedimentation for preparing media with limited inorganiccarbon. The following buffers were used at 5 mol m~3: pH 7.5HEPES/Ca(OH)2 or NaOH, or pH 10 CAPS/NaOH or CAPS/Ca(OH)2.

Dark techniques—When using Synechococcus, dark experi-ments must be run in completely dark conditions (Ritchie 1991). Adim, green safe-light was only used for taking samples (about 10 s,0.1 /rniol (quanta) m~2 s"1 PAR).

Membrane filtration techniques—A Millipore filtration ap-paratus with a sintered glass surface was used for filtration assays(Ritchie and Gibson 1987, Ritchie 1991). Uptake of MRb+ wasmeasured using filtration techniques as previously described (Rit-

chie et al. 1996) using Nuclepore® (Costar Corp., Cambridge.MA, U.S.A.) polycarbonate membrane filters (0.4 or 1 /mi). Theusual specific activity was =300 to 10,000 GBq mol"1.

The cell wall of Synechococcus binds significant amounts ofcations (Ritchie and Gibson 1987, Ritchie 1992a). Cells were wash-ed with 20 mol m~3 Ca(NO3)2 (buffered to pH 7.5 with HEPES/CaJOH)^ for about 10 s to remove most of the extracellularMRb+. Cells were exposed to ^Rb* radiolabel for a few seconds,were washed and then used as a measure of binding of 86Rb+ tothe surface of the cells. Binding of 32PO4 to cells was much slowerthan for 86Rb+, taking about 1 min to reach steady-state, and wasmuch more difficult to wash from the cells.

Inorganic phosphate—Total reactive inorganic phosphatewas assayed using a semi-micro version of the molybdate/stan-nous chloride method described in APHA (1992). The intensemolybdenum blue colour produced by the reduction of themolybdophosphoric acid by stannous chloride was measured at700 run. Standard curves were linear to 50 mmol m~3 PO4.

Soluble phosphate in experimental cell suspensions was usual-ly estimated by centrifuging in Eppendorf tubes in a Beckman Mi-cro fuge (6,000 x g) and sampling the supernatant. Cell densities ofabout 600 x 1012 cells m"3 were used.

Intracellular phosphate—To estimate intracellular phos-phate, a cell suspension was filtered onto a polycarbonate filterand washed in buffered calcium nitrate. The cells were then sus-pended in an Eppendorf tube in water containing chloroform (1%v/v) for 10 min then centrifuged and the supernatant assayed forreactive soluble phosphate.

Uptake ofnP— Both chemical assays and 32P assays of phos-phate showed that significant amounts of phosphate bind to thesurfaces of Synechococcus cells (Falkner et al. 1980). Saturationkinetics were only observable at concentrations below 5 mmolm~3 using very dilute cell suspensions (A75o=0.2=60x 1012 cellsm~3) and short labelling times. 32P uptake was measured using 0.2to 5 mmol m~3 phosphate (specific activity = 100 x 109 to 13 x 1012

Bq mol"1) and incubation periods of 2 min and 5 min. The re-sidual phosphate in the "P-free" incubation media was meas-ured and taken into account in specific activity calculations ( = 0.1to 4 mmol m"3). Cells were pre-incubated for one h in bufferedphosphate-free BG-11 before commencing a labelling experiment.32P labelled phosphate was added to the cap of the incubationvials (Eppendorf tubes) and labelling' started by shaking. At thetwo minute time point, 0.5 ml of the cell suspension was removedfrom an incubation vial and filtered onto a 0.4 //m filter and wash-ed twice with unlabeled BG-11 to remove as much surface bound32P-label as possible. The cells on the filter were then resuspendedin 0.5 ml of distilled water, 3 ml of scintillant was added and thesamples were counted. Fluxes were calculated from the differencein uptake by the cells after the 2 and 5 minute incubation times asmol m~2 s"1. At shorter labelling times it was not possible to dis-tinguish intracellular uptake from the extracellular bound compo-nent because binding of phosphate to the cells was slower than inthe case of cations.

O2 evolution—Photosynthesis was measured using a Clark-type electrode (Hansatech, Kings Lynn, Norfork, U.K.) ther-mostatted to 25°C and used as described by Walker (1990). Theelectrode was calibrated with aerated water for 100% air satura-tion and with N2 as zero O2. The oxygen solubility algorithms ofCarpenter (1966) and Colt (1984) were used to calculate the oxy-gen concentration in air-saturated medium. The chamber was illu-minated with saturating red light (peak 660 nm), provided by alight emitting diode array (175 ^mol (quanta) m"2 s"1 PAR).

Harvested cells (~300x 1012cells m"3) were filtered onto

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1234 Phosphate uptake in a cyanobacterium

polycarbonate membrane niters, then washed three times in experi-mental media. Oxygen electrode experiments were run on cells in-cubated for about 1 h in phosphate-free BG-11 at pHo7.5 andpre-incubated in the light. Samples were purged with water saturat-ed N2 gas before 1 ml of cell suspension was placed in the oxygenelectrode chamber. The effect of 50 mmol m~3 phosphate wastested using a paired t-test (control vs. added phosphate).

Membrane potential—The membrane potential (A !Pi,0) wasmeasured using the valinomycin expedited MRb+ equilibrationtechnique described and discussed in Ritchie (1991) and Ritchie etal. (1996) but using filtration and washing to measure Valino-mycin-mediated uptake of s*Rb+.

The membrane potential was calculated from the accumula-tion ratio of the permeant cation (Ritchie 1991, 1992a) using theNernst equation (Nobel 1983) after cell wall binding was correctedfor as described above. Activities were calculated from concentra-tions using the Bjerrum version of the Debye-Huckel equation(Hamer 1968).

For consistency with another study, all A fu0 estimates usingthe 86Rb+/valinomycin probe quoted in the present paper are bas-ed on 2 h, rather than 1/2 h incubations.

Transitory polarization of the membrane potential—Provid-ed a cation is taken up passively (86Rb+/valinomycin) and its per-meability across the cell membrane is constant, the uptake rate ofthe cation under a control and experimental condition can be usedto detect polarizations and recoveries of A ¥, o (Ritchie 1992a, Rit-chie et al. 1996).

The A Yx_0 of the experimentally treated cells can be calculatedas;

Equation 1

where, F/RT are the Faraday, Gas Constant and absolute tempera-ture respectively, A !Pe is the membrane potential of cells given theexperimental treatment at t=0 , A *FC is the membrane potential ofthe control cells (assumed to be constant), <t>c is the uptake flux ofthe permeant cation in the experimentally treated cells, & is theflux of the permeant cation in the control cells. Equation 1 canonly be solved iteratively. The error of A ^ can be estimated by apartial differentiation with respect to A Vz, <j>c and 0C (Young 1962).

Equilibrium accumulation of these permeant cations wasroutinely measured for both control and experimental treatmentsand so both the initial effects of an offered substrate and the mem-brane potential after the cells had been exposed to the changedconditions for a considerable time could also be measured.

Intracellular pH—Intracellular pH (pHJ was determined us-ing silicone gradient centrifugation techniques as described in Rit-chie (1991) and Ritchie et al. (1996). The weak acid, I4C-DMO(5,5-dimethyloxazoline-2,4-dione), was used as the pH-probe forthe experiments at pHo 7.5 and the weak base, 14C-methylamine,for pH: determinations at pHo 10. Both probes were used at a finalconcentration of 100 mmol m~3 with an incubation time of about5 min. In the 14C-methylamine experiments, 100 mmol m~3 NH«"was present to discourage metabolism of methylamine. Label car-ried through the silicone oil with the cells in the extracellular waterspace and label bound to the surfaces of the cells were allowed for.

Kinetics—Uptake of phosphate was followed routinely for upto 6 h. Net uptake vs time was fitted to an exponential model us-ing a non-linear least squares fit (Johnson and Faunt 1992) givingestimates of the exchange constant (k) and initial concentration(E0,t=.o). and the initial uptake flux calculated as k-Eo l = 0 . TheKm

and V^a of the isotopic uptake flux of 32P were estimated usingnon-linear least squares fitting with at least ten data points in each

experiment (Ritchie and Prvan 1996a, b). ±95% confidence limitswere then calculated using n-2 degrees of freedom (Zar 1974).

Counting methods—32P and 8<Rb+-label were counted usingthe "P-channel of a Canberra-Packard Tri-CARB 1600 scintilla-tion counter using Emulsifier-Safe scintillant.

Statistics—Error-bars are ±95% confidence limits with thenumber of replicates in brackets 0- Where two numbers appearin brackets (a, b), the first is the number of separate experimentsconducted and the second is the total number of observations.Students t-tests, linear regressions including the error-limits of theslopes (m) and y-intercepts, and other statistics were calculated asdescribed by Zar (1974).

Results

Uptake of phosphate—Figure 1A shows the uptake ofphosphate by Synechococcus cells versus time in the light.After the 30 min pre-incubation period in nominally phos-phate-free medium the cells maintained an extracellularphosphate of about 1 mmol m~3. Cell suspensions didnot pull extracellular phosphate down to zero. Followingthe addition of 50 mmol m~3 phosphate, the cells rapidlydepleted the phosphate to near zero over 4 h. It was ex-pected that such a curve could be used as a method forestimating the Km of phosphate uptake (Duggleby 1985).Uptake follows an exponential decay curve which impliesthat the net flux is proportional to concentration. Figure IBshows a plot of the rate of uptake of phosphate by Synecho-coccus cells versus time, calculated as the rate of uptake be-tween each sampling time, using the data from Figure 1A.The net uptake rate was a linear function of concentrationand does not exhibit saturation kinetics (Fig. 1 A, B). The y-intercept is not significantly different to zero (—0.208 ±0.225) and so the rate of uptake is directly proportional toconcentration (0PO4=k«[PO4]o; r=0.990, where k=41.6(±5.8)x 10~'ms"'). The saturable component of phos-phate uptake was only apparent if very low phosphate con-centrations were used (below 5 mmol m~3; see Methods:Uptake of 32P).

Preliminary experiments showed that phosphate up-take was strongly inhibited at acid pH0, had a broad max-imum at pHo 7 to 9 then fell off at more alkaline con-ditions. Uptake of phosphate was significantly higher(ANOVA p<0.001) in the light than in the dark, and therate of uptake of phosphate was much greater at pH 7.5than at pH 10 in the light (ANOVA p<0.001). Under thealkaline conditions under which the cells were grown,pH 0 ~ 10, the net uptake rate could be described by theequation (0PO4=k-rPO4]o, where k=9.0 (±1.8)xl0~ 9

ms"1). pH did not significantly alter the rate of phosphateuptake in the dark (Table 1). The photosystem II inhibitor,DCMU (5 mmol m~3), decreased the rate of phosphate up-take by the cells in the light from 1,770±240 (6) pmol m~2

s~' to l,270±370 (6)pmolm~2s""' (p=0.02) and so didnot decrease net phosphate uptake to the very low ratesfound in the dark.

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|a.

1 ..s•2

[PO4]O = 55.12*10"0.326t

r = 0.999

• Extracellular phosphate

Q Preincubated zero PO4

ato

5a•5X3E2s

= 0.047[PO4] - 0.208

r = 0.990

• Net PhotphaM Flux(nmol m-2 »"1)

OJ 1 1.5

Time (h)

Fig. 1A

1 S 2 0 2 5 3 0 1 5 4 0 4 S 5 0 5 5 M

External PO4 (mmol m"3)

Fig. IB

Fig. 1A Uptake of phosphate by Synechococcus cells versus time in the light. Cells were pre-incubated in the experimental conditionsfor 30 min before the addition of phosphate. The cells had reduced the extracellular phosphate to about 1 mmol m~3 during the pre-in-cubation period in nominally phosphate-free medium. The pH0 was 7.5 using 5 mol m~3 HEPES/Ca(OH)2 buffer. Care was taken toadd enough Na2CO3 so that cells did not run out of inorganic carbon before the incubation was complete. Uptake follows an exponentialdecay curve.

Fig. IB Rate of uptake of phosphate by Synechococcus cells versus time in the light using the data from Fig. 1A calculated as the rateof uptake between each sampling time shown in Fig. 1 A. The net uptake rate is a linear function of concentration and does not exhibit

Phosphate uptake rates of cells growing in continuousculture could be calculated from the upstream and down-stream concentrations of phosphate, the washout rate andthe cell numbers in the effluent stream (Tempest 1970).Table 1 shows that the phosphate consumption rate of theexponentially growing cells (=600 pmol m~2 s"1) was close-

ly comparable to that measured on harvested cells un-der similar conditions (pH0= 10). The effluent stream con-tained 18±3 (12,44) mmol m"3 from a feedstock of =50mmol m~3 and so were not phosphate limited cells.

Table 2A shows the intracellular phosphate concentra-tions of the alga after 1 h incubation with either nominally

Table 1 Phosphate

Condition

consumption by Synechococcus

Net uptake rateLight

(pmolrn~2s"')Dark

Harvested cells

pHo7.5

pH0 10

Continuous culture

(~pHo 10)

l,303±146 (11,118)

449± 90(11,83)

586± 65 (12,48)

136±91 (5,41)

146±53 (5,38)

NA

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Table 2A Thermodynamic data for analysis of phosphate uptake

[P04]0(mraol ra"3)

Phosphate inmedia and cells

POJ,(mol m )

Membrane potential(mV)

Intracellular pH ila or protonmotive force(kJmor1)

Light Dark Light Dark Light Dark Light Dark

pH07.5 1.7310.64 5.9+1.9 3.010.8(9) (5,20) (2,8)

pH07.5 50 9.9+2.4 9.713.3 -13211.2 -11411.6 7.3810.12 7.0010.18 -13.410.7 -13.911(5,20) (3,14) (2,16) (2,8) (3,12) (3,12)

pHolO 3.8112.08 7.711.5 4.712.2(5) (3,12) (2,8)

pHo10 50 10.311.5 8.911.2 -12912.8 -13113.1 7.2210.07 7.3610.11 +3.410.5 +2.410.11(3,12) (3,14) (7,44)' (5,20)° (6,36)* (4,24)*

" Combined data from this study and Ritchie et al. (1996).* Taken from Ritchie et al. (1996).

zero (about 1 to 4 mmol m 3) or 50 mmol m 3 added phos-phate. Under low-phosphate conditions the intracellularphosphate concentration is about halved in the dark atboth pHo 7.5 and 10. For cells in low phosphate the ap-parent intracellular phosphate was 6.6± 1.3 mol m~3 (8,32)or 787 ±173 nmolm" 2 on a cell surface area basis in thelight. Cells in 50 mmol m~3 phosphate had a higher intracel-

, lular phosphate concentration of 10±1.6molm~ 3 (8,32)or 1.21 ±0.22//mol m~2. There was no apparent differencein intracellular phosphate in the light or dark or for cells inpHo 7.5 or 10 when high external phosphate was present.

Chemically detectable free phosphate was only a smallproportion of the total phosphate accumulated by the cells.The chemostat data could be used to calculate the totalphosphorus held by the cells from the depletion of phos-phate from the medium and the volume of the cells in theeffluent stream. The total intracellular phosphorus was424±47 mol m" 3 (12,48). Thus, only about 2A±0A% oftotal intracellular phosphorus is present as phosphate.

Table 2B Thermodynamics of phosphate uptake at pH0 7.5

Intracellular pH and membrane potential—Synecho^coccus cells are able to maintain their intracellular pH with-in narrow limits, even when the cells are maintained at apHo which is several pH units different to that of the inter-nal concentration (Ritchie 1991, Ritchie et al. 1996). Cellsincubated at pHo 10 have similar membrane potentials andintracellular pH in the light and dark. Cells incubated inthe dark at pH0 7.5 have a different membrane potentialand intracellular pH to those of cells incubated at pH0 7.5in the light. The membrane potential and intracellular pHof cells in the light at pH0 7.5 are similar to those found inboth the light and dark at pH0 10. Using the A !PiiO and pHjdata, the electrochemical potential for protons or protonmotive force (A/xH^0 or pmfi>0) was about —13 kJ mol"1 atpH 0 7.5 (active extrusion of H + ) but only about + 3 kJmol"1 at pH0 10 (active uptake of H + ) .

Thermodynamic analysis—Tables 2B and 2C presentthe results of thermodynamic analyses, using the Nernstcriterion, and show the driving force required to active-

Species

H2PO4"HPO4~PO3,-Total

H2PO4"HPO4~PO3,"Total

[PO4]Omol m 3

500 ±185 xlO"6

1.23± 0.46 xlO"3

20 ± 7.4 xlO"9

1.73+ 0.64X10"3

14.4 xlO"3

35.6 xlO"3

577 x10"9

50 x 10"3

Light[POJli

molm"3

1.95 ±0.653.95±1.31

48±16xlO"«5.9 ±1.9

3.27±0.846.63 ±1.69

80±21xl0" 6

9.9 ±2.4

AuPOlikJ mol"^

+ 33 + 1.2+45 ±0.63+ 57±0.43

+26±0.65+ 38±0.34+ 50±0.24

Dark

[PO4]imolm

1.63 ±0.451.38+0.38

6.9±1.9xlO"6

3.0 ±0.8

5.26±1.844.45 ±1.56

22±7.9xlO"6

9.7 ±3.3

AfiPOzti

kJ mol+ 31±1.15+ 39±0.60+47±0.41

+ 25±0.88+ 33±0.46+ 41 ±0.33

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Table 2C Thermodynamics of phosphate uptake at pH0 10

Species

H2PO4"HPO4~POJ-Total

H2PO4"HPO2"PO4"Total

mol m"3

4.9 ± 2.7 x lO- 6

3.79± 2.07 xlO"3

19 ±11 xlO"6

3.8 ± 2.1 xlO"3

64 x 10-6

49.7 xlO"3

255 x 10-6

50 x 1Q-3

Light

[PO4]jmolm"3

3.20±0.643.50±0.90

38±7.5xlO- 6

7.7 ±1.5

4.29±0.656.02±0.91

5O±7.6xlO-6

10.3 ±1.5

kJ m o r 5

+45±1.5+42±0.77+ 38±0.55

+40±0.46+ 36±0.33+ 33±0.3

Dark

[PO4Lmolm 3

1.60±0.763.10±1.47

36±17xlO"6

4.7 ±2.2

3.03±0.465.87±0.90

68±10xl0" 6

8.9 ±1.2

kJ mor°+44±1.82+41 ±0.95+ 39±0.67

+ 39±0.46+ 37±0.33+ 34±0.32

ly transport each species of soluble phosphate into thecell (Nobel 1983). The relative amounts of each form ofphosphate was calculated using the equations and pKa

values published by Lindsay (1979). No form of phosphate(HjPOr, HPO4~ or PO4~) could be accumulated passivelyby the cells. All forms are at least +25 kJ mol"1 from elec-trochemical equilibrium at both pH07.5 and 10.

Uptake of 32P—At pH07.5 the Km was 1.65±1.04(4,44) mmol m~3 and 2.92± 1.46 (4,47) mmol m"3 at pHo

10. These values were not significantly different and so anoverall mean of 2.31 ±0.90 (8,91) mmol m~3 could be cal-culated. Vmax was also similar at pH0 7.5 (KmM=3.52±0.52(4,44) nmol m ^ s " 1 ) and pHo 10 (Vmlx=4.25±1.04 (4,47)nmol m~2 s~l) giving an overall mean of 3.90±0.59 (8,91)nmolm~2s~'. Isotopic fluxes were considerably higherthan the chemically measured net fluxes and showed differ-ent kinetics.

Taking the intracellular phosphate content on a cellsurface area basis calculated above (=1.2/«nol m~2) andthe observed isotopic flux, the exchange constant is 3.22(±0.761) x l 0 ~ 3 s " ' o r a tI/2 of 215±51 s.It can be conclud-ed that the isotopic flux is a measure of phosphate trans-port across the plasmalemma, whereas the chemical assay,because of the time course used, is a measure of metabolicuptake of phosphate.

O2 evolution—Synechococcus has a very large intracel-lular store of phosphorus and so short-term incubations inphosphate-free media was not expected to affect photosyn-thesis or respiration. There was no significant effect of add-ed phosphate in the dark using a paired t-test or non-parametric sign test (R= 12.6± 1.55 nmol m~2s~' (3,12)).There was, however, a significantly inhibitory effect uponphotosynthesis of added phosphate when it was addedto the phosphate-free cells (paired t-test, p=0.2%; Non-parametric Sign test p=0.02%). The gross photosyntheticrate (Po) of the control cells was 56.2±4.7 nmol m~2s~'(3,12) vs. 50.9±3.6 nmol m"2 s~' (3,12) after adding phos-phate. These R and PG rates are comparable to those found

previously for Synechococcus (Ritchie et al. 1996).Effects of ion deficiencies—The effects of different ions

upon phosphate uptake rates are shown in Table 3. Lack ofK+ decreased phosphate uptake by about 50%. Phosphateuptake in nominally Na+-free media was consistently in-hibited by approximately 80%. Nominally Na+-free mediacontained about 6 mmol m~3 Na+ due to contamination ofchemicals (Ritchie et al. 1996). Lack of Ca2+ had no effecton the uptake of phosphate by Synechococcus. Mg24" notonly decreased the net uptake of phosphate, but there wasa net loss by the cells. The effects of lack of Mg2"1" appearedto be irreversible as adding this ion back into a cell suspen-sion in Mg2+-free BG-11 did not lead to a recovery of phos-phate uptake by the cells.

Electrogenicity of phosphate uptake—Table 4 showsthe polarizing effects of added phosphate (50 mmol m~3)measured using the Valinomycin-mediated uptake of 86Rb+

as the membrane potential probe (Equation 1). The effectsof phosphate uptake upon the membrane potential appearto be different at pHo 7.5 and 10. This is consistent with thegreat differences in the abundances of HzPO^ and HPO2"at pH0 7.5 and 10 (see above). At pH0 7.5 uptake of phos-phate in the light led to a hyperpolarization of the apparentmembrane potential. This indicates a net uptake of nega-

Table 3 Effect of mineral ion deficiencies on the uptakeof PO4 in the light at pH0 10

IonUptake flux (pmol m 2 s ')

With added cation Cation omitted

K+

Na+

Ca2+

Mg2+

+ 546±70

+ 871 ±276

+ 308±71

+426±121

(2,12)

(5,39)

(4,27)

(1,7)

+280 ±47

+ 174±40

+ 361 ±159

-145 ±47

(2,12)

(5,37)

(2,13)

(2,13)

Fluxes are positive for net uptake and negative for net loss by thecells.

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Table 4 Polarization effects of phosphate using 86Rb+/valinomycin as the membrane potential probe

Conditions

BG-11, pHo7.5, LightBG-11, pHo7.5, DarkBG-11, pH07.5, Light+DCMUBG-11, pHo10, LightBG-11, pHo10, DarkBG-11, pHo 10, Light+DCMU

A'F,control

-132±2-112±1.8-122±1.8-128±4.6-126±1.8-137±1.9

Membrane potential (mV)

120 min exposure

-132±2-115±1.8-128±1.9-128±5.1-129±1.9-137±2

AVccpolarization

- 5 9 ±23 (2,64)- 1 7 ± 1 8 (32)- 2 6 ± 8 (32)+ 39± 7.7 (3,96)- 4 ± 1 8 (32)

+ 50± 9.7 (32)

Cells were pre-incubated in BG-11 medium in capped Eppendorf tubes. The valinomycin-mediated uptake flux of !686+ was measured at0, 2, 4 and 6 min. Control and experimental cells were incubated as matched pairs. The ^Rb"1" fluxes were measured by shaking the tubesand removing a cell sample for filtration after the appropriate labelling time. Membrane potentials were calculated from the equilibriumaccumulation ratio of ^Rb* after labelling for 120 min of both control and experimentally treated cells.

tive charge when the cells were supplied with phosphate. Asimilar, but smaller effect, was found in the presence ofDCMU but there was no apparent polarization effect in thedark. This result is consistent with findings on the effects ofDCMU and darkness on phosphate uptake. At pHo 10, inthe light with and without DCMU there was a depolariza-tion, not hyperpolarization of the membrane potential. Asat pH0 7.5, there was no apparent effect in the dark. In allcases, any electrical effect of phosphate is transitory: after a120 minute incubation there is only a small or no differencein the membrane potential of cells with or without phos-phate.

Discussion

Distinguishing transport of phosphate across theplas-malemma from assimilation of phosphate—At concentra-tions of phosphate above about 5 mmol m~3, net uptakewas directly proportional to concentration with no satura-ble component (Fig. 1A, B) and if uptake flux was plottedagainst concentration, the apparent intercept was throughzero. Wagner et al. (1995) found that when phosphate-starved cells were subjected to pulses of phosphate, the up-take behaviour of the cells was linearised, but failed to rec-ognize that the y-intercept passed through zero. Since thecells used in the present study were not phosphate-limited,then the linearized behaviour found in the present studyand by Wagner et al. (1995) is not restricted to phosphatelimited cells.

Isotopic fluxes were considerably higher than the chem-ically measured net fluxes and showed saturable kinetics iflabelling times comparable to the filling time of the intracel-lular inorganic phosphate pool (t , / 2~4min) and very lowphosphate concentrations were used. The Km for phos-phate was only about 2 mmol m~3 and the saturating fluxwas about 4 nmol m~2 s"1. The Km value is comparable toprevious estimates in Synechococcus (Budd and Kerson

1987, Avendano and Valiente 1994, Wagner et al. 1995),other cyanobacteria (Istvanovics et al. 1993, Valiente andAvendano 1993) and the bacterium Streptococcus (Pool-man et al. 1987). Taking the intracellular phosphate poolon a surface area basis, it was possible to calculate aturnover time for the cytoplasmic phosphate. Turnover ofthe intracellular chemically detectable phosphorus was veryrapid (ti/2~215±51 s) which is comparable to an estimateof = 10 min by Suttle et al. (1991). Thus net uptake experi-ments, such as Figure 1, conducted over a period of hours,with samples taken every 15 or 30 min, were actually meas-uring the rate of incorporation of phosphate into polyphos-phate storage material.

Kinetic analyses of phosphate uptake in Synechococ-cus have shown that both the Km and Vmax are not signifi-cantly different at pH0 7.5 and pH010. This has the implica-tion that the phosphate uptake mechanism operating inneutral media is the same as that operating at alkaline pH0.From the abundance of H2POi" and HPOj" at pHo 7.5 andpH0 10, both the mono- and divalent species of phosphateare accepted by the transport system. At pH0 7.5 about29% of total phosphate is in the form of H2PO4~ and 71%in the form of HPOl~ whereas at pHo 10 only 0.13% ispresent as H2PO4~, 99.4% as H?Ol~ and 0.5% as PO3,"(Lindsay 1979).

Active uptake of phosphate—The process of metabol-ic assimilation of phosphate by Synechococcus is energy de-pendent is indicated by the substantial decrease in the rateof uptake when the experiments were performed in thedark. This result agrees with the results for some species ofcyanobacteria (Avendano and Valiente 1994), but does nothold true for all species (Istvanovics et al. 1993). Phosphateuptake is not significantly decreased in the presence of thephotosynthetic inhibitor DCMU, indicating that ATP isthe likely energy source for active uptake of phosphate.Similar conclusions have previously been drawn for Cl~(Ritchie 1992b, c), HCO3" (Ritchie et al. 1996) and S O ^

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(Ritchie 1996).Thermodynamic analysis clearly demonstrates that

phosphate is actively taken up by the cells regardless ofwhich form of phosphate is considered (Table 2B, C). Allforms of phosphate are at least +25 kJ moP1 from equilib-rium and so a driving force of at least —25 kJ moP1 isrequired to drive active uptake of phosphate. Tables 2Aand 2B however, show that a proton-motive-force (pmfio)driven mechanism is very unlikely in Synechococcus. Thepmfi0 at pH0 7.5 is only about - 1 3 kJ moP1 but at pH0 10the pmfio is positive, about 2 to 3 kJ moP1. At pH0 10 pro-tons are actively taken up by the cells and is far short of the+ 3 0 k J m o P ' required for active uptake of phosphate(Table 2B). A pmfio-driven pump is conceivable at pH0 7.5but would need to have a stoichiometry of at least 4H + /PO4 (4 x —13 kJ moP1) but such a mechanism would notbe thermodynamically possible under alkaline conditions.

Effect of ion deficiencies—The rate of phosphate up-take was significantly reduced when the cells were incubat-ed in Na+-free media, as found previously by Avendanoand Valiente (1994) and for CP transport (Ritchie 1992b,c) and HCO3~ transport (Ritchie et al. 1996) but contrastingwith the lack of a Na+ effect in the case of SOJ~ transport(Ritchie 1996). Avendano and Valiente (1994) also found aK+-effect comparable to that found in the present study.Comparison of Tables 1 and 3 show that the net phosphateuptake in Na+-free media in the light fell to the rates typi-cally found in the dark.

In contrast to the present study, Budd and Kerson(1987) reported that Na+ was not required for phosphateuptake in Synechococcus; a result quite different to thatfound in the present study and by Avendano and Valiente(1994). Rigby et al. (1980) and Budd and Kerson (1987) alsotested for the effects of K+, Ca2+ and Mg2+ upon phos-phate uptake by cyanobacteria. Rigby et al. (1980) andBudd and Kerson (1987) both reported a stimulatory effectof Ca2+ but not Mg2+. However, their results were proba-bly not reliable, as inappropriate experimental media wereused for their experiments. They first isolated their cells inTris/H2SO4 buffer (pH 8) then tested for the effects of add-ed cations. Not only is Tris known to be toxic to cyanobac-teria but the Tris/H2SO4 buffer probably caused irreversi-ble damage to the cells because of the lack of magnesiumions. We found that the damaging effect of Mg2+-freemedia was not readily reversible. Washing the cells inmagnesium-free media was enough to disable the mechan-ism for phosphate uptake. Table 3 shows Ca2+ had no ap-parent effect on phosphate uptake. Since the Mg2+-freeBG-11 used in the present study contained Ca2+, K+ andNa+ it can be concluded that none of these cations can sub-stitute for Mg2+. Lack of Na+ or K+ inhibited phosphateuptake but since K+-free BG-11 contained Na+, apparentlyNa+ will not substitute for K+.

Polarization studies—Significant transient polariza-

tion effects of added phosphate were detected in the lightbut not in the dark (Table 4) despite the inherent impreci-sion of using valinomycin expedited uptake of 86Rb+ tomeasure changes in membrane potential (Ritchie 1992a, Rit-chie et al. 1996). DCMU did not abolish the polarizationeffects of added phosphate in the light. At pHo 10, wherevirtually all phosphate is present as HPO2~ (Table 2C),added phosphate depolarizes the membrane potential in-dicating a net uptake of positive, not negative charge. AtpHo7.5, where substantial H2PO4~ is present (Table 2B),added phosphate hyperpolarizes the membrane potential.Complex charge balancing operations are involved in ac-tive uptake of phosphate.

Mechanism of uptake of phosphate—Poolman et al.(1987) suggested that uptake of phosphate in Streptococcuswas electroneutral, with a variable number of protons be-ing cotransported with the phosphate depending on thevalency of the phosphate being transported. Table 4 doesnot point to an electroneutral uptake mechanism.

Tables 2A, 2B and 2C show that a proton-motive-force-driven mechanism is very unlikely in Synechococcus.The pmfi0 at pH0 7.5 is only about - 1 3 kJ moP1 (Table2B) but at pH0 10 the pmfio is positive, about 2 to 3 kJmoP ' Table 2C). At pH0 10, protons are actively taken upby the cells and the pmfio is far short of the —30 kJ moP 1

required for active uptake of phosphate (Table 2C). Apmfio-driven pump would not be thermodynamically possi-ble under alkaline conditions.

Table 3 shows that phosphate uptake was clearly Na+-dependent as found previously for CP and HCOf andother ions but this does not necessarily point to the elec-trochemical potential for Na+ (4^Na£,) being the drivingforce for active uptake of phosphate (Ritchie 1992b, c, Rit-chie et al. 1996). Avendano and Valiente (1994) found noeffect of Na+ upon the Km of phosphate uptake but theyfound a large effect on the rate of uptake. The AfiNa£0 forSynechococcus cells in the light is about —13 to —16 kJmoP1 at pHo7.5 and 10 (Ritchie 1992a, Ritchie et al.1996). At least 4Na+ would be needed to drive the uptakeof one phosphate (Table 2B, C). Such an uptake mechan-ism would be strongly electrogenic leading to uptake of netpositive charge for each phosphate taken up at both pH0

7.5 and 10, contrary to the complex polarization effectsshown in Table 4.

Falkner et al. (1980), Budd and Kerson (1987) andothers have proposed that phosphate uptake by Synecho-coccus is an ATP-driven primary transport system. The hy-drolysis of an ATP molecule produces a driving force of ap-proximately - 5 0 kJ mol"1 (Reid and Walker 1983, Muchland Peschek 1984) and so the phosphate uptake mechan-ism would need to have a stoichiometry of one phosphatetaken up per ATP to be thermodynamically possible underall the conditions tested in the present study. For thermody-namic reasons a 1 PO4/ATP pump could not reduce exter-

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nal phosphate concentrations to much below the 1 mmolm~3 found for cells incubated in nominally P-free media.Freshly made up P-free media had an inorganic phosphateconcentration well below 0.1 mmol m" ' and so cells lostphosphate until reaching a steady-state between uptake andloss. Table 2A suggests that the cells are unable to maintaintheir intracellular phosphate in the dark under low phos-phate conditions because phosphate uptake is so stronglylight-dependent. The stalling potential of the phosphate up-take mechanism is about —40 kJ mol"1 or a thermodynam-ic efficiency of about 80%.

Compared to the magnitude of the fluxes of other ionssuch as sodium (Ritchie 1991, 1992a) and bicarbonatefluxes (Ritchie et al. 1996) both the isotopic and net fluxesof phosphate are very low and so not likely to impose alarge metabolic cost to the cells. If it is assumed that oneATP is used to take up one phosphate, then the power con-sumption of a normal phosphate uptake flux (Table 1)would be 5 8 6 ± 6 5 p m o l m ~ 2 s " ' x 5 0 k J m o r ' = 0 . 0 2 9 ±0.003 mW m~2 (Ritchie et al. 1996). This power consump-tion is very small compared to the requirements of Na+ andHCO^" transport and the power available from photosyn-thesis (Ritchie 1992a, Ritchie et al. 1996). Not unexpected-ly, oxygen electrode experiments have failed to findlarge effects on respiration or photosynthesis of addedphosphate to cells prepared in P-free media in the shortterm.

Phosphate requirements of exponentially growingSynechococcus—The progressively changing nutrient en-vironment of a batch culture is not a realistic model for analgal-bloom being continuously fed new nutrients from thestream-flow. Chemostats can be used as laboratory mod-els of cyanobacterial blooms in streams receiving treatedeffluent from sewage treatment plants (Tempest 1970).

Table 1 shows that Synechococcus consumes about600pmolm~2s~' of phosphate to sustain optimum ex-ponential growth in a medium with a phosphate concen-tration comparable to the effluent of a typical secondarysewage plant (50 mmol m~3). In practical situations, an ex-ponentially growing Synechococcus culture is capable of as-similating nearly all phosphate offered to it. However, thecells are not capable of pulling dissolved phosphate downto undetectable levels because at about 1 mmol m~3 exter-nal phosphate an ATP-driven pump is close to its thermo-dynamic limits in maintaining intracellular phosphatelevels (Table 2B, C). In continuous culture, Synechococcushas an intracellular pool of total phosphate equivalent to400 mol m~3: this store of phosphorus would be able tosupply cells with adequate phosphate for about 6 genera-tions or at least 3 days. Cyanobacteria are notorious forscavenging phosphate and storing it as polyphosphatewhich takes several cell generations to deplete after environ-mental phosphate has been exhausted (Falkner et al. 1989).

Genetic database of Synechocystis—The complete se-

quence for one cyanobacterium, Synechocystis PCC 6803is on the INTERNET as a genetic database called CYA-NOBASE (bibliography; http://www.pka3.kazusa.or.ip/cyano/biblio.html, similarity search; http://www.kazusa.or.ip/cyanobase/kwd.html). It has a very large listing ofputative ion transporters including phosphate transporters.The mechanism of action of some of these transporters hasbeen identified from sequence homologues (Kaneko et al.1996a, b). For example, one phosphate transporter hasbeen identified as a primary ABC-type ATP-driven pump(pstB: sllO683; sll0684; slrl250). Two other phosphate trans-porters are known (pstA: sllO682; slrl249 and pstC:sll0681; sir 1248) whose mechanism of action are unknown.Periplasmic phosphate binding proteins have also beenidentified, one of which is associated with phosphate starva-tion (slrl247). The present physiological findings on Syne-chococcus are consistent with current genetic informationon cyanobacteria.

Dr. R.J. Ritchie holds an ARC Postdoctoral Research Fellow-ship. 32P-H3PO4 was purchased from the Australian Institute ofNuclear Science and Engineering (AINSE). Dr. Donelle Trautmanwas supported in part from an ARC Institutional Grant awardedto Dr R.J. Ritchie and Prof A.W.D. Larkum.

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(Received June 16, 1997; Accepted September 4, 1997)

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