Research Phosphate limited cultures of the cyanobacterium Synechococcus are capable of very rapid, opportunistic uptake of phosphate Blackwell Science Ltd Raymond J. Ritchie1, Donelle A. Trautman2 and A. W. D. Larkum2 1 Science, Food & Horticulture K-12, University of Western Sydney-Hawkesbury, Richmond NSW 2753, Australia; 2Biology A-08, School of Biological Sciences, The University of Sydney, NSW 2006, Australia. Summary Author for correspondence: R. J. Ritchie Tel:+61 24570 1436 Fax: +61 24570 1621 Email: r.ritchie@uws.edu.au Received: 29 January 2001 Accepted: 18 July 2001 • Phosphate uptake rates were measured in Synechococcus R-2 incubated in artificial secondary- and tertiary-treated sewage. • Phosphate uptake was measured using chemical assay and 32P incorporation. Intracellular pH was measured using accumulation of 14C-labelled weak acids and bases and membrane potentials using 86Rb+/valinomycin. • Synechococcus cells are capable of very rapid, opportunistic uptake of phosphate (10–30 nmol m−2 s−1) even though net uptake by growing cultures was < 0.5 nmol m−2 s−1. Km and Vmax in the light were not significantly different at pHo 7.5 and 10. The mean Km values were 1.91 ± 0.41 mmol m−3 and 0.304 ± 0.055 mmol m−3 for P-sufficient (secondary-treated sewage) and P-deficient (tertiarytreated sewage) cells, respectively. The transport systems probably recognize both H2PO4− and HPO42−. Intracellular inorganic phosphate is +28 to +56 kJ mol−1 from electrochemical equilibrium. In P-sufficient cells uptake is very slow in the dark (c. 0.1 nmol m−2 s−1) but phosphate-starved cells can opportunistically take up P about 100 times faster. • Two separate ATP-driven phosphate uptake mechanisms (1 PO4 in per ATP) appear to be responsible for phosphate uptake by the cells. They have different Km values, different light/dark responses and electrical behaviour. Key words: Cyanobacteria, sewage, phosphate uptake kinetics, P-limited cells, P-unlimited cells, ammonia-N, nitrate-N. Abbreviations ASS, artificial secondary sewage; NOXAS, artificial tertiary sewage; CAPS, 3[Cyclohexylamino]-1-propane-sulphonic acid; DMO, 5,5-dimethyloxazoline2,4-dione; c, refers to the control-treated cells; e, refers to the experimental treatment cells; i, refers to the inside of the cells; o, refers to the outside of the cells or bulk electrolyte; ∆ψi,o is the membrane potential measured from the outside to the inside. © New Phytologist (2001) 152: 189–201 Introduction Phosphate is often blamed for causing cyanobacterial blooms but its removal from wastewater is very expensive, and phosphate might not be directly responsible for blooms. A © New Phytologist (2001) 152: 189–201 www.newphytologist.com predictive model system is needed to test water treatment strategies and water management policies. The cyanobacterium (blue-green alga) Synechococcus R-2 is a good model organism for many membrane transport studies (Ritchie & Gibson, 1987; Ritchie et al., 1996; Ritchie, 1998). 189 190 Research Recently our laboratory made a study of the thermodynamics and uptake kinetics of phosphate in Synechococcus grown in a nitrate-based medium with luxury amounts of phosphate (Ritchie et al., 1997). The routinely used growth medium for many studies of nutrient transport by Synechococcus is BG-11 medium (Allen, 1968). BG-11 was developed as a storage medium for cyanobacteria and was not intended to mimic environmental conditions under which cyanobacteria occur in nature. For example, BG-11 has very high concentrations of NaNO3 (18 mol m−3) and 0.175 mol m−3 PO4. Such concentrations are very unlikely to be met in the field. Despite the work of Kromkamp et al. (1989) on chemostatgrown Microcystis, little is known about the physiology of uptake of phosphate by cyanobacteria under conditions likely to be encountered in the environment. Unfortunately, the strains of Anabaena and Microcystis often responsible for toxic cyanobacterial blooms have thick mucilaginous sheaths, which make mechanistic membrane transport studies on them very difficult or impractical. Synechococcus is more suitable for mechanistic studies of nutrient uptake. Synechococcus has been used in several studies of severe phosphate stress (Grillo & Gibson, 1979; Falkner et al., 1980; Mohleji & Verhoff, 1980; Falkner et al., 1984; Budd & Kerson, 1987; Falkner et al., 1989; Wagner et al., 1994; Falkner et al., 1995). This study provides information on the mechanism of uptake of phosphate in media formulated to closely resemble the effluent of typical secondary sewage treatment and tertiary treatment facilities. The major form of nitrogen in secondary sewage is ammonia-N (as dissolved NH3 + NH4+) at concentrations usually exceeding 1 mol m−3. Tertiary sewage treatment facilities are often called NOx Plants because they convert the ammonia-nitrogen in secondary-sewage largely to N2 gas and the small amount of fixed-N that remains in the form of nitrate. Additionally, phosphate is decreased to very low concentrations by chemical precipitation (as alum, calcium or iron salts). Secondary and tertiary treated effluent are therefore very different in respect of the nitrogen source available and the concentrations of available phosphate. In this study, Synechococcus R-2 has been grown using continuous culture methods to simulate the behaviour of a cyanobacterial bloom receiving a continuous input of nutrients from either a secondary or a tertiary treatment plant. Materials and Methods Chemicals and radiochemicals 86Rb+, 14C-ethanolamine and 14C-methylamine were obtained from DUPONT New England Nuclear, Boston, MA, USA, 14C-DMO from CEN SACLAY-Molecules Marqees 91191 GIF-sur-Yvette, France and 32P-H3PO4 from the Australian Nuclear Science Technology Organization (ANSTO), Lucas Heights, Sydney, Australia. DCMU, DMO, HEPES, ethanolamine, methylamine and valinomycin were purchased from Sigma-Aldrich Chemical Company, St Louis, MO, USA. All reagents used were of analytic standard. Culture material Synechococcus R-2 (S. leopoliensis, Anacystis nidulans) (strain PCC 7942), was grown from axenic stock cultures in BG-11 medium (Allen, 1968) modified as described previously (Ritchie & Gibson, 1987). Intracellular volumes, cellular surface areas and numbers of cells were calculated from measurements of light scattering (A750 nm) using a Varian® Cary 13E UV-Visible spectrophotometer (Varian Australia, Melbourne, Victoria, Australia) (Ritchie & Gibson, 1987; Ritchie, 1991). General methods Cultures were routinely grown in a continuous culture device, in continuous light of about 150 µmol (quanta) m−2 s−1 PAR, using cool white fluorescent lights, at 30°C and supplied with filtered air. Semi-continuous culture was not satisfactory because the cells tended to deplete the medium of phosphate and become phosphate-limited. The pHo of the reactor effluent of a culture growing in either Artificial Secondary Sewage (ASS) or Artificial Tertiary Sewage ( NOXAS) was about 10 –10.5. Cells were usually harvested by centrifugation (2500 g) in a preparative centrifuge and washed three times in the experimental medium. The following buffers were used at 5 mol m−3: pH 7.5 HEPES/Ca(OH)2 or HEPES/NaOH; pH 10 CAPS/ NaOH or CAPS/Ca(OH)2. TRIS buffers were avoided because TRIS is toxic to Synechococcus (Rigby et al., 1980). Low cell densities (about 30–60 × 106 cells/ml) were used in the experiments to avoid depleting the phosphate too quickly. A routine preincubation of 30 min was run in the light or dark as appropriate, before using cells in the experiments. Experiments were run at 25°C under conditions of illumination similar to those used for growing the cells or in the dark as appropriate. ASS medium contained 2 mol m−3 inorganic carbon, which would not have been depleted by the cells during incubation in the present study. Enough Na2CO3 (1 mol m−3) was added to the NOXAS medium so that cells did not run out of inorganic carbon before the incubation was complete. In cultures that are strongly aerated and in equilibrium with air or 5% (v/v) CO2 the pH of the medium is approx. 7.5. When cultures are gently stirred and not fully in equilibrium with the air the steady-state pH is approx. 10. Therefore, pH 7.5 and pH 10.5 were chosen for the present study. Uptake rates for phosphate were measured in the light and dark. Phosphate depletion was measured using the standard acid molybdate assay or by 32P-labelling (Ritchie et al., 1997; American Public Health Association (APHA), 1998). All glass and plastic laboratory-ware were washed in dilute HCl before use. The steady-state intracellular phosphate concentration of cells growing in the chemostat could be estimated by treating www.newphytologist.com © New Phytologist (2001) 152: 189–201 Research effluent cell suspensions with chloroform (for 1 min), followed by centrifugation (6300 g for 1 min), then assaying the total inorganic phosphate in the supernatant. The extracellular phosphate concentration of centrifuged controls, not treated with chloroform, was then subtracted. Methods used for the measurement of the uptake of 32P (including techniques for running radiolabelling experiments in the dark, methods for membrane filtration and inorganic phosphate assays), membrane potential and transitory polarization of the membrane potential have all been described previously (Ritchie, 1991; Ritchie et al., 1997; Ritchie, 1998). Radioactivity was counted using a Canberra-Packard TriCarb 1600 scintillation counter (Packard Instruments, Meriden, CT, USA) (Ritchie et al., 1997). Artificial secondary sewage medium (ASS) The artificial secondary treated sewage medium (ASS) was based upon typical analyses of the effluent of a sewage treatment plant ( Warriewood STP) in the Sydney metropolitan area, NSW, Australia (Table 1). The plant uses a secondary activated sludge process and the effluent can be taken as representative of effluent produced by such plants (American Public Health Association (APHA), 1998). No attempt was made to Table 1 Typical analyses of a representative effluent from a secondary and a tertiary treatment plant and artificial secondary sewage (ASS) and artificial tertiary sewage media (NOXAS) Na+ K+ NH4+ Mg2+ Ca2+ Cl− HCO3− HPO42− SO42− NOx (all forms) Trace elements (mmol m−3) Fe Cu Zn Mn devise a trace element mix similar to the sewage because the concentrations of trace elements in the sewage were broadly similar to the concentrations found in the trace element mix used in BG-11 medium (Allen, 1968). Synechococcus grew well in ASS medium, though in batch culture it tended rapidly to deplete the medium of phosphate. In continuous culture the pHo of the reactor effluent of a growing culture was approx. 10–10.5. Phosphate-free ASS medium contained < 0.1 mmol m−3 phosphate. In ASS medium, fixed nitrogen is predominantly present as ammonia rather than nitrate. The potassium, magnesium, calcium and sulphate concentrations are very similar to those in BG-11. The sodium content (Table 1) is somewhat lower than in BG-11 medium (18 mol m−3 Na+), but would easily fulfil the well-documented sodium requirements of Synechococcus (Ritchie et al., 1996; Ritchie, 1998; Ritchie, 1991, 1992a,b,c). At 50 mmol m−3, the phosphate concentration of ASS was also lower than that of BG-11 (c. 175 mmol m−3). Artificial tertiary sewage medium (NOXAS) The artificial tertiary treated sewage medium (NOXAS) was based upon typical analyses (Table 1) of the effluent of tertiary sewage treatment plants (Sydney Water). Secondary treated effluent (mol m−3) ASS medium (mol m−3) Tertiary treated effluent (mol m−3) NOXAS medium (mol m−3) 5.66 0.44 2.00 0.700 0.450 5.36 1.98 0.053 0.521 0.024 5.68 0.44 2.00 0.627 0.450 5.67 2.00 0.050 0.050 0.024 5.66 0.44 0.060 0.700 0.450 5.36 0.200 0.003 0.521 0.350 5.86 0.44 0.0 0.500 0.450 6.2 0.200 0.003 0.500 0.350 8.95 1.57 3.06 1.82 8.95 1.57 3.06 1.82 Artificial Secondary (ASS) and Artificial Tertiary (NOXAS) Sewage Culture Media Salts NaCl KCl NH4HCO3 Na2HPO4 NaHCO3 CaCl2 MgSO4 NaNO3 ASS mol m−3 5.56 0.44 2.00 0.05 0 0.45 0.50 0.024 pHo 7.7, use trace element mix as for BG-11 medium. © New Phytologist (2001) 152: 189–201 www.newphytologist.com NOXAS mol m−3 5.30 0.44 0 0.003 0.200 0.45 0.50 0.350 191 192 Research In NOXAS medium, fixed nitrogen was predominantly present as nitrate but at a concentration much below that found in BG-11 medium and close to that found in eutrophic waters. Phosphate-free NOXAS medium contained < 0.1 mmol m−3 phosphate. Synechococcus grown in NOXAS medium were distinctly yellow-green in colour and could be shown to be phosphate limited. The phosphate content of NOXAS medium was very low (3 mmol m−3) and hence was closely similar to that likely to be found in eutrophic natural waters (American Public Health Association (APHA), 1998). Intracellular pH Intracellular pH (pHi) was determined using the weak acid, for experiments at pHo 7.5, or the weak bases, 14C-methylamine and 14C-ethanolamine, for pH 10 (Ritchie o et al., 1996). All probes were used at a final concentration of 100 mmol m−3 with an incubation time of approx. 5 min. Since ASS medium contained 2 mol m−3 NH4HCO3, sufficient ammonia-N was present to discourage metabolism of methylamine. In experiments using NOXAS medium, 100 mmol m−3 ammonia-N was added to discourage metabolism of methylamine. 14C-DMO, Membrane potentials and polarization effects of phosphate uptake Membrane potentials (∆ψi,o) were measured using the 86Rb+/ valinomycin technique described previously (Ritchie, 1991; Ritchie et al., 1996; Ritchie et al., 1997) using 5 mmol m−3 valinomycin. The transient polarization effects caused by transport of added substrate (in this case phosphate) were measured as described previously (Ritchie et al., 1997). To measure the polarization effects of different experimental conditions, Synechococcus cells were first preincubated in phosphate-free ASS medium or NOXAS medium in capped Eppendorf tubes. The valinomycin-mediated uptake flux of 86Rb+ was then measured at 0, 2, 4 and 6 min. Control and experimental cells were incubated as matched pairs. For the phosphate treatment, sufficient phosphate for a final concentration of 50 mmol m−3 was placed in the cap, along with 86Rb+/valinomycin. The 86Rb+ fluxes were measured by shaking the tubes and removing a cell sample for filtration after the appropriate labelling time. Membrane potentials were calculated for both control and experimentally treated cells from the equilibrium accumulation ratio of 86Rb+ after labelling for 30 min. Statistics Kinetics For the kinetic experiments, cells were centrifuged and suspended in nominally phosphate-free ASS or phosphatefree NOXAS medium with the appropriate buffer added. Uptake was measured using chemical assay of phosphate or 32P. In the chemical assay method, disappearance of phosphate from the medium was followed by measuring the phosphate in the supernatant after pelleting the cells by centrifuging in a Beckman Microfuge (Beckman Instruments, Palo Alto, CA, USA) (about 6300 g for 1 min). Loss of phosphate was exponential with time. Nonlinear curve fitting methods (Johnson & Faunt, 1992) were used to estimate the rate constant (k), initial concentration (Eo,0) and the initial uptake flux (calculated as k.Eo,0). Phosphate uptake could also be followed using 32PO4 and silicone-oil centrifugation as previously described (Ritchie et al., 1997). Uptake vs time was fitted to an exponential saturation model. Asymptotic uptake (Ei,∞) and the rate constant were estimated by nonlinear least squares fitting and the initial uptake flux calculated as k.Ei,∞. In kinetic experiments it was found that uptake of phosphate by the cells was approximately linear for up to 120 s. Binding of phosphate to the cell surface was corrected for by measuring uptake of 32P by cell suspensions that were labelled for as short a time as practicable then centrifuged (total labelling time about 2 s). The Km and Vmax of the isotopic uptake flux of 32P were estimated using nonlinear least squares fitting (Ritchie & Prvan, 1996). The model allowed for substrate inhibition. Error-bars are ± 95% confidence limits with the number of replicates shown in brackets. Where two numbers appear in brackets (a,b), the first is the number of separate experiments conducted and the second is the total number of observations. Other statistics were calculated as described by Zar (1974). Results Experimental material Synechococcus cells grown in ASS medium had a doubling time of 7.7 ± 0.67 (12) h, which is considerably faster than that of 12.1 ± 1.44 (24) h, found in cells grown in nitrate-based media (Ritchie et al., 1997). Synechococcus thus grows much more rapidly with ammonia as the nitrogen source. However, for reasons that are not clear, cultures grown at pH 10 were found to die off after several days if not provided with trace amounts of nitrate (23 mmol m−3; Table 1). Synechococcus grown in the NOXAS medium had a doubling time of 23.4 ± 1.99 (12) h, based upon cultures drawn from the reactor, fed 3 mmol m−3 PO4 and incubated for 12 h. The doubling time based upon the turnover time of the reactor was 25.8 ± 0.63 (42) h. When the cells were given fresh medium with 50 mmol m−3 phosphate and incubated for 12 h, they had a doubling time of 9.99 ± 2.6 (6) h. Thus, cells grown in NOXAS medium were clearly phosphate-limited. www.newphytologist.com © New Phytologist (2001) 152: 189–201 Research Table 2 Thermodynamic data for analysis of phosphate uptake for cells in artificial secondary sewage (ASS) and artificial tertiary sewage media (NOXAS) medium ASS cells Membrane Potential (mV) pHo 7.5 pHo 10 Intracellular pH (pHi) pHo 7.5 pHo 10 ∆µH+i,o or pmfi,o (kJ mol−1) pHo 7.5 pHo 10 NOXAS cells Membrane Potential (mV) pHo 7.5 pHo 10 Intracellular pH (pHi) pHo 7.5 pHo 10 ∆µH+i,o or Proton Motive Force (kJ mol−1) pHo 7.5 pHo 10 Membrane potential and intracellular pH The differences in the composition of ASS and NOXAS medium had little effect on the membrane potentials (∆ψi,o) of the cells (Table 2). However, the membrane potential was consistently less negative in the dark compared with the light. Cells incubated in the dark at pHo 7.5 and 10 had similar membrane potentials but the ∆ψi,o in the light at pHo 7.5 was more negative than that found at pHo 10. Synechococcus cells are able to keep their intracellular pH within narrow limits, even when maintained at a pHo that is several pH units different from that of pHi (Ritchie, 1991; Ritchie et al., 1996; Ritchie et al., 1997). Estimates of pHi shown in Table 2 for the cultures grown in ASS medium are closely comparable to those reported previously for nitrategrown cells (Ritchie et al., 1997) even though, at pHo 10, most of the ammonia would have been present as NH3. Synechococcus is thus unusually tolerant of high concentrations of free ammonia (NH3). The estimate of pHi for cells at pHo 10 in the light, using the 14C-ethanolamine probe, was 7.49 ± 0.10 (4,24), which was not significantly different from estimates made using 14C-methylamine (pHi = 7.47 ± 0.08 (8,32) ). The data were combined in Table 2. The intracellular pH of the cells grown in NOXAS medium was significantly more acid than for cells grown in ASS medium and also lower than that found previously in Synechococcus cells grown in BG-11 medium (Ritchie, 1991; Ritchie et al., 1996; Ritchie et al., 1997). Apparently, limited phosphate supply interferes with regulation of intracellular pH. Using the ∆ψi,o and pHi data, the electrochemical potential for protons or proton motive force (∆µH+i,o or pmfi,o) could be calculated. At pHo 7.5 the pmfi,o was approx. –10 kJ mol−1 indicating active extrusion of H+ in the light and the dark. © New Phytologist (2001) 152: 189–201 www.newphytologist.com Light Dark −147 ± 1.4 (4,16) −133 ± 4.1 (5,20) −124 ± 5.4 (4,16) −122 ± 1.1 (5,20) 7.39 ± 0.089 (4,16) 7.48 ± 0.062 (12,56) 6.93 ± 0.193 (4,16) 7.43 ± 0.031 (3,18) −13.6 ± 0.5 +1.6 ± 0.5 − 8.7 ± 1.2 +2.9 ± 0.3 −155 ± 3.5 (3,12) −148 ± 2.1 (3,12) −132 ± 6.4 (3,12) −138 ± 2.2 (3,12) 6.78 ± 0.12 (6,24) 7.20 ± 0.028 (3,12) 6.88 ± 0.112 (6,24) 7.16 ± 0.035 (3,12) −10.8 ± 0.8 +1.7 ± 0.3 −9.2 ± 0.9 +2.9 ± 0.3 On the other hand the pmfi,o was very near zero (approx. +2 to +3 kJ mol−1) at pHo 10 indicating active uptake of H+. Under the alkaline conditions under which the cells were grown, there was very little or no pmfi,o available to drive secondary active transport. Uptake of phosphate Uptake rate of steady-state continuous cultures Rates of phosphate uptake for cells growing in continuous culture could be calculated from the upstream and downstream concentrations of phosphate, the washout rate and the cell numbers in the effluent stream (Ritchie et al., 1997). Phosphate consumption by cells supplied with ASS medium in continuous culture was 400 ± 41 (25 100) pmol m−2 s−1. The effluent stream contained a substantial portion (22 ± 7.6 (11) mmol m−3) of the phosphate supplied as a feedstock (approx. 50 mmol m−3). The cells were not phosphatelimited. The phosphate consumption rate of cells fed NOXAS medium in continuous culture was much lower than that of P-sufficient cells (98 ± 14 (42) pmol m−2 s−1). The effluent stream contained 1.03 ± 0.192 (6,36) mmol m−3 phosphate from a feedstock of approx. 3 mmol m−3 so the cells were not able completely to deplete the external phosphate. The cells were clearly phosphate-limited (see above). Uptake of phosphate by cells in ASS medium Fig. 1 shows the uptake of phosphate by Synechococcus cells in ASS medium (pHo 10) in the light, dark and in the presence of DCMU (photosystem II inhibitor). After preparing the cells in P-free ASS-medium, then preincubating for 30 min, the cells were offered 50 mmol m−3 phosphate and the disappearance of phosphate from the medium was monitored every 5 min. 193 194 Research Fig. 1 Uptake of phosphate by P-sufficient cells of Synechococcus grown on ammonia-N in the light (open sqauares), dark (closed squares) and in the presence of 5 mmol m−3 DCMU in the light (circles). In this experiment the loss of phosphate from the medium was measured by chemical assay. Cells were preincubated for 30 min, in nominally phosphate-free artificial secondary sewage (ASS) medium, before addition of phosphate. The cells had decreased the concentration of extracellular phosphate to approx. 1 mmol m−3 during the preincubation period (diamond, bottom left). The pHo was maintained at 7.5 using 10 mol m−3 HEPES/Ca(OH)2 buffer. Uptake followed exponential decay curves: φin is the initial rate of uptake of phosphate as mol m−2 s−1 calculated from the fitted exponential curves. When uptake flux was plotted against concentration, the apparent intercept coincided with zero. Thus net uptake was directly proportional to concentration, with no saturable component. After a 30-min preincubation period in nominally phosphate-free ASS medium, the extracellular phosphate concentration had decreased to approx. 1 mmol m−3. In the light, uptake of phosphate was much faster than measured previously (Ritchie et al., 1997). Following addition of 50 mmol m−3 phosphate, the cells rapidly depleted phosphate to approx. 1 mmol m−3 after only 30 min. Remaining phosphate decreased exponentially with time, showing that the uptake Fig. 2 Uptake of phosphate (32P-label) by Synechococcus in artificial tertiary sewage (NOXAS) medium in the light (open sqaures) dark (closed squares) in the presence of 5 mmol m−3 DCMU in the light (circles). The dotted line shows the point at which all added phosphate would have been removed from the medium. Cells were preincubated for 30 min before addition of phosphate in nominally phosphate-free NOXAS medium. The cells had decreased the extracellular phosphate to approx. 1 mmol m−3 during this preincubation period. The pHo was maintained at 10 using 10 mol m−3 CAPS/Ca(OH)2 buffer. Uptake followed an exponential curve. rate was directly proportional to the external concentration. The initial uptake rate was approx. 24 nmol m−2 s−1. A depletion curve following this shape cannot be used to estimate the Km of the uptake mechanism (Duggleby, 1985). Uptake rates in the dark were very slow compared to the rates found in the light and were difficult to measure using chemical assays. Nevertheless, the rate of uptake rapidly fell to near zero after about 30 min. The photosynthetic inhibitor, DCMU, only partially inhibited the uptake of phosphate in the light (Valiente & Avendano, 1993). Uptake of phosphate by cells in NOXAS medium Fig. 2 shows the uptake of phosphate by Synechococcus cells in the light, dark and in the presence of DCMU using 32P at pHo 10. www.newphytologist.com © New Phytologist (2001) 152: 189–201 Research Table 3 Thermodynamics of phosphate uptake (a) at pHo 7.5 in ASS medium, (b) at pHo 10 in ASS medium, and (c) at pHo 10 in NOXAS medium Light Species (a) (b) (c) [PO4z]o mol m−3 Dark [PO4z]i mol m−3 ∆µPO4zi,o kJ mol−1 [PO4z]i mol m−3 ∆µPO4zi,o kJ mol−1 H2PO4− HPO42− PO43 − Total 14.4 × 10 −3 35.6 × 10 −3 577 × 10 −9 50 × 10 −3 5.51 ± 0.84 11.45 ± 1.74 142 (± 22) x 10 − 6 16.96 ± 2.10 28.4 ± 0.40 42.0 ± 0.23 55.6 ± 0.19 12.19 ± 2.66 8.77 ± 1.91 38 (±8.3) x 10 − 6 20.96 ± 2.12 28.2 ± 0.75 36.9 ± 0.59 45.6 ± 0.55 H2PO4− HPO42− PO43 − Total 63.8 × 10 − 6 49.7 × 10 −3 255 × 10 − 6 50 × 10 −3 3.64 ± 0.43 9.31 ± 1.09 142 (±17) x 10 − 6 12.95 ± 1.28 39.5 ± 0.62 37.7 ± 0.41 36.2 ± 0.43 11.7 ± 1.19 26.6 ± 2.7 361 (± 37) x 10 − 6 38.22 ± 3.70 41.5 ± 0.58 38.4 ± 0.16 35.5 ± 0.14 H2PO4− HPO42− PO43− Total 3.94 × 10 − 6 2.981 × 10 −3 15.4 × 10 − 6 3 × 10 −3 1.72 ± 0.184 2.31 ± 0.247 18.5 (±1.98) x 10 − 6 4.03 ± 0.426 45.9 ± 0.33 44.2 ± 0.24 42.5 ± 0.22 5.99 ± 2.19 7.21 ± 2.64 51.4 (±18.8) x 10 − 6 13.2 ± 4.83 47.6 ± 0.93 44.3 ± 0.50 41.0 ± 0.37 After a 30-min preincubation period in nominally phosphatefree NOXAS medium, the extracellular phosphate concentration was approx. 1 mmol m−3. Following addition of 5 mmol m−3 phosphate, the cells rapidly depleted the phosphate to near zero after only 30 min. Previous kinetic experiments had shown that the Km of phosphate uptake was well below 1 mmol m−3, so 5 mmol m−3 should have saturated the uptake rate of phosphate. The initial uptake rates were 34 ± 5.8 nmol m−2 s−1 n = 11 (light), 24 ± 3.8 nmol m−2 s−1 n = 11 (+ DCMU, light) and 8.7 ± 2.9 nmol m−2 s−1 n = 11 (dark) which are very high for ion fluxes in Synechococcus. DCMU only partially inhibited the uptake of phosphate in the light (as found in ASS-grown cells). Similar results were found for Synechococcus cells incubated at pHo 7.5. Overall, the saturating phosphate uptake rate was found to be 35 ± 3 nmol m−2 s−1 (4,44, light), 28 ± 7 nmol m−2 s−1 (12, + DCMU, light) and 8.2 ± 1.2 nmol m−2 s−1 (4,44, dark) at pHo 7.5 and 30 ± 3 nmol m−2 s−1 (4,44, light), 24 ± 4 nmol m−2 s−1 (12, + DCMU, light) and 8.7 ± 2.9 nmol m−2 s−1 (4,44, dark) at pHo 10. Intracellular phosphate Monitoring of phosphate uptake by continuous cultures could also be used to calculate the intracellular concentration of P. Chemically detectable free phosphate was only a small proportion of the total P accumulated by the cells. For the ASS-grown cells, the total intracellular P was 341 ± 26 mol m−3 (15,60). The estimate of intracellular reactive phosphate from cells in continuous culture was 12.3 ± 1.8 (6,24) mol m−3, which is closely comparable to estimates of [PO4]i made on incubated cell suspensions (Table 2). Thus, only about © New Phytologist (2001) 152: 189–201 www.newphytologist.com 3.6 ± 0.5% of total intracellular phosphorus was present as phosphate in cells growing in the chemostat. At pHo 7.5, the intracellular phosphate concentration in Synechococcus cells, incubated for 1 h in ASS medium with 50 mmol m−3 added phosphate, was slightly greater in the dark than in the light (P < 0.01; Table 3a,b). However, at pHo 10, intracellular phosphate was very different in the light and dark. In the light, intracellular phosphate was about 13 mol m−3 (not greatly different from that found at pHo 7.5) whereas in the dark it was about 38 ± 4 mol m−3. Since phosphate uptake by the cells is virtually zero after prolonged incubation in the dark, this very much greater intracellular phosphate must arise from mobilization of intracellular polyphosphate in darkness. Total intracellular P for cells grown in a reactor supplied with NOXAS medium was only 48 ± 4.2 mol m−3 (6,36): much below that found for P-sufficient cells (ASS medium, above) or found previously in cells well supplied with phosphate and nitrate in BG-11 medium (Ritchie et al., 1997). After removal from the reactor and incubation in NOXAS medium with 3 mmol m−3 added phosphate for 1 h at pHo 10, cells had an intracellular reactive phosphate of 4.03 ± 0.426 (4,24) mol m−3 in the light and 13.2 ± 4.83 mol m−3 (3,24) in the dark (Table 3C). Only approx. 8 ± 1% of total intracellular P is present as phosphate in cells growing on NOXAS medium at an external pHo of about 10. Thermodynamic analysis Tables 3a, b and c present the results of thermodynamic analyses, using the Nernst criterion, and show the driving force required actively to transport each species of soluble 195 196 Research phosphate into the cell (Nobel, 1983). The membrane potential values were taken from Table 2 and estimates of activities were made using the Debye-Huckel equation. The concentrations of each form of phosphate inside and outside the cells were calculated using the equations and pKa values published by Lindsay (1979). The electrochemical potential (in kJ mol−1) was then calculated for each phosphate ion separately using the Nernst equation. No form of phosphate (H2PO4−, HPO42− or PO43−) could be accumulated passively by the cells under any of the conditions used in this study. All forms are at least +28 kJ mol−1 from electrochemical equilibrium for cells in ASS medium in both the light and dark. In NOXAS medium, which had a very much lower phosphate concentration, all forms are at least +40 kJ mol−1 from electrochemical equilibrium at pHo 10 in the light and dark. Uptake kinetics of 32P At pHo 7.5, using cells grown in ASS medium, there is some evidence for substrate inhibition at high phosphate concentrations but the apparent Km is low, approx. 2 mmol m−3 (Fig. 3). The overall mean of Km determinations run at pHo 7.5 was 1.70 ± 0.75 (4,63) mmol m−3 and 2.10 ± 0.58 (4,72) mmol m−3 at pHo 10. These values were not significantly different and so an overall mean of 1.91 ± 0.41 (8135) mmol m−3 could be calculated. Vmax was also similar at pHo 7.5 (12.5 ± 2.05 (4,63) nmol m−2 s−1) and pHo 10 (12.1 ± 1.92 (4,72) nmol m−2 s−1) giving an overall mean of 12.3 ± 1.38 (8135) nmol m−2 s−1. Attempts to measure the Km in the dark were not successful. Uptake was so low and difficult to measure accurately, that uptake vs concentration curves could not be fitted to a Michaelis-Menten model. For NOXAS-grown cells, the apparent Km was very low, < 1 mmol m−3. Km and Vmax were estimated using a nonlinear least-squares fit model that allowed for substrate inhibition. Loading times of 0.5 or 1 min were used. The overall mean of Km determinations was 0.31 ± 0.096 (5,65) mmol m−3 at pHo 7.5 and 0.29 ± 0.083 (4,54) mmol m−3 at pHo 10. These values were not significantly different and so an overall mean of 0.304 ± 0.0552 (9119) mmol m−3 could be calculated. Vmax was very high at both values of pHo but significantly different: at pHo 7.5, Vmax was 32.4 ± 1.5 (5,65) nmol m−2 s−1 and at pHo 10, Vmax was 20 ± 0.70 (4,54) nmol m−2 s−1. Estimates of sustained uptake of phosphate using 32PO were 0.14 ± 0.08 (5,68) nmol m−2 s−1 at pH 7.5 and 4 o 0.11 ± 0.03 (5,42) nmol m−2 s−1 at pHo 10. These rates are closely comparable to those based upon phosphate depletion by continuous cultures growing on NOXAS medium (see above). The phosphate-starved cells grown in NOXAS-medium were able to take up phosphate in the dark, at rates comparable to those found in the light. At pHo 7.5, Vmax = 7.9 ± 0.9 (13) nmol m−2 s−1 with a Km of 0.25 ± 0.17 mmol m−3 and at pHo 10, Vmax = 11.6 ± 1.8 (13) nmol m−2 s−1 with a Km of 0.69 ± 0.43 mmol m−3. The intracellular phosphate content of cells grown in ASS medium, calculated on a volume basis (Table 2), could be recalculated on a cell surface area basis (1.47 ± 0.260 µmol m−2) using conversion factors from Ritchie (1991). Taking the observed isotopic flux at saturating external phosphate and the intracellular phosphate pool size, the intracellular phosphate pool would be completely replaced in 125 ± 25 s. Similar calculations for cells grown in NOXAS show that the pool is very small (0.482 ± 0.070 µmol m−2). Such cells, if suddenly offered 5 mmol m−3 phosphate in the light, are capable of taking it up at 30 nmol m−2 s−1. The intracellular phosphate pool would be completely replaced in only 16 s in the light. For the uptake rates shown in Fig. 2 to be sustainable, the cells must have been converting imported phosphate into storage material at a very rapid rate. Electrogenicity of phosphate uptake Fig. 3 Effect of phosphate concentration on the uptake of 32PO4 by Synechococcus in the light. Cells were preincubated in phosphatefree artificial secondary sewage (ASS) medium at pHo 7.5 for 30 min before addition of phosphate. Uptake rates were based upon measurements during in the case of both P-limited and P-unlimited cells the first 2 min after adding 32P-labelled phosphate. The fitted line gives a value for the Km of 1.49 ± 0.72 mmol m−3 and a Vmax of 26.3 ± 4.4 nmol m−2 s−1. Inhibition by high phosphate concentrations could be described by −0.21 (± 0.11) µm s−1 × [P]. Table 4 shows the polarizing effects of added phosphate. For cells grown in ASS, phosphate was added at 50 mmol m−3. The effects of phosphate uptake on membrane potential appear to be different at pHo 7.5 and 10. At pHo 7.5, uptake of phosphate in the light led to a hyperpolarization of the apparent membrane potential. At pHo 10, in the light there was a depolarization, not hyperpolarization, of the membrane potential. In the case of cells grown in NOXAS medium, the polarizing effects of 5 mmol m−3 added phosphate were measured. At pHo 7.5, uptake of phosphate in the light led to a very large www.newphytologist.com © New Phytologist (2001) 152: 189–201 Research Table 4 Polarization effects of phosphate using 86Ru/valinomycin as the probe of membrane potential Membrane potential (mV) Conditions ∆ψi,o Control ∆ψi,o 30 min exposure ∆ψe,c Polarization ASS, pHo 7.5, Light ASS, pHo 10, Light NOXAS, pHo 7.5, Light NOXAS, pHo 10, Light −160 ± 3 −161 ± 2.5 −157 ± 2.0 −154 ± 2.3 −158 ± 2.6 −162 ± 2.6 −163 ± 2.7 −160 ± 3.3 − 41 ± 17 (2,48) +29 ± 8 (2,48) −133 ± 15 (3,48) −52 ± 12.2 (3,48) hyperpolarization of the apparent membrane potential but the effect was much smaller at pHo 10. Any electrical effect of the rapid uptake of phosphate is transitory: after incubation for 30 min there was no difference in the membrane potential of cells with or without added phosphate. Discussion Distinguishing transport of phosphate across the plasmalemma from assimilation of phosphate In our previous study of nitrate-grown Synechococcus (Ritchie et al., 1997), little difference could be found between cells grown as batch (1/4 dilution each day) and continuous cultures. However, the cells used in this study were grown in lower phosphate concentrations and tended rapidly to deplete the media of phosphate. In continuous culture, cells grown in ASS medium were not phosphate-limited because the concentration of phosphate in the reactor never approached the Km of the uptake mechanism (approx. 2 mmol m−3). Cells grown in NOXAS medium depleted the extracellular phosphate to near the thermodynamic stalling point of the uptake mechanism and were demonstrably P-limited. Only cells grown in continuous culture were used in the present study. Fig. 1 shows that Synechococcus depleted ASS medium of phosphate at a rate that was directly proportional to the concentration present in the incubation medium and hence the rate of uptake decreased exponentially over time. Similar behaviour was noted previously for BG-11-grown cells (Ritchie et al., 1997). The initial uptake rate (appox. 25 nmol m−2 s−1) was very high for ion fluxes in Synechococcus. The linearized behaviour found in the present studies and by Wagner et al. (1995) and Falkner et al. (1995) is not restricted to phosphate limited cells. Classical saturable kinetics of phosphate uptake (Fig. 3) was found in ASS and NOXAS-grown cells only if very short labelling times were used. Saturable kinetics was apparent only if labelling times were comparable to the filling time of the intracellular inorganic phosphate pool (t1/2 appox. 1 min). The Km for phosphate of ASS-grown cells was approx. 2 mmol m−3 and the mean saturating flux was approx. 12 nmol m−2 s−1. The Km value is comparable to previous © New Phytologist (2001) 152: 189–201 www.newphytologist.com estimates in Synechococcus grown in various media (Rigby et al., 1980; Budd & Kerson, 1987; Avendano & Valiente, 1994; Wagner et al., 1995; Ritchie et al., 1997), other cyanobacteria (Kromkamp et al., 1989; Istvanovics et al., 1993; Valiente & Avendano, 1993) and the bacterium Streptococcus (Poolman et al., 1987). The Km for phosphate uptake in P-limited cells grown in NOXAS medium was much lower than found in P-sufficient cells, both in the present study and in our previous study (Ritchie et al., 1997). The cells were capable of taking up phosphate very rapidly in the light (approx. 30 nmol m−2 s−1) when offered phosphate but were also capable of taking up phosphate at a significant rate in the dark as well. The Km was very low (approx. 0.3 mmol m−3): much lower than found in P-sufficient cells. By recalculating the intracellular phosphate pool on a surface area basis, it was possible to calculate a filling time for the cytoplasmic phosphate. The filling time for ASS-grown (P-sufficient) cells was approx. 125 ± 25 s but was only apporx. 16 s for P-limited cells. The residence time of PO4 inside Synechococcus cells is thus very short (Rigby et al., 1980; Ritchie et al., 1997). This has important implications when interpreting phosphate uptake experiments. Net uptake experiments, such as those shown in Figs 1 and 2, with samples taken on a scale of minutes, actually measure the rate of incorporation of phosphate into polyphosphate storage material rather than the cellular uptake step. Kinetic analyses of phosphate uptake in Synechococcus have shown that both Km and Vmax were not significantly different at pHo 7.5 and pHo 10 but that there appeared to be a different uptake mechanism operating under P-sufficient and P-limited conditions. P-sufficient cells, whether using ammonia-N (ASS – this study) or nitrate-N (BG-11, Ritchie et al., 1997) as fixed nitrogen sources, appear to express the same phosphate uptake mechanism, based upon the Km and light/ dark behaviour. P-limited cells appear to use a different mechanism with a much lower Km which, unlike the low-affinity mechanism, is capable of operating at a high rate in the dark. Nevertheless, from the abundance of H2PO4− and HPO42− at pHo 7.5 and pHo 10 (Table 3a,b), both the mono- and di-valent species of phosphate must be accepted by both transport systems (Lindsay, 1979). The kinetic experiments shown in Figs 1 and 2 necessarily involved preparing the cells in phosphate-free ASS or NOXAS 197 198 Research medium. The uptake rates of such cells were many times higher (> 10 nmol m−2 s−1) than found in cells in steady-state in continuous culture. Harvesting and washing the cells by centrifugation took approx. 1 h and was performed largely in dim light (approx. 5 µmol m−2 s−1); the cells were then preincubated in standard light conditions for 30 min. It seems likely that the capacity of ammonia-grown cells to take up phosphate at very high rates is constitutive rather than induced. Cells incubated in phosphate-free medium for several hours did not increase their rate of phosphate uptake (5 min 32P-PO4 assay) by more than 50%. Active uptake of phosphate In both P-limited and P-unlimited cells, there is a significant light/dark effect on assimilation of phosphate by Synechococcus. Phosphate uptake was not greatly decreased in the presence of the photosystem II inhibitor DCMU (Fig. 1), indicating that ATP is the likely energy source for active uptake of phosphate. Uptake of phosphate is strongly light dependent. Furthermore, the results using DCMU show that fixed carbon is not needed for either uptake by the cells or its rapid storage as insoluble polyphosphate. The thermodynamics of phosphate uptake in Synechococcus cells grown with ammonia-N as the nitrogen source are essentially similar to those in nitrate-grown cells (Ritchie, 1991; Ritchie et al., 1997). Thermodynamic analysis clearly demonstrates that phosphate is actively taken up by the cells, regardless of which form of phosphate is considered (Table 3). Tables 2 and 3 however, show that a pmfi,o-driven mechanism is very unlikely in Synechococcus grown in ASS or NOXAS medium. The pmfi,o at pHo 7.5 is only about −13 kJ mol−1 but at pHo 10 the pmfi,o is positive, about +2 to +3 kJ mol−1. At pHo 10, protons are actively taken up by the cells. The pmfi,o is far short of the +28 kJ mol−1 required for active uptake of phosphate (Table 3). In line with previous findings (Ritchie et al., 1997) there was a consistent increase in detectable inorganic phosphate inside the cells in the dark compared to the concentrations found in the light (see Table 3). However, net uptake of phosphate was found to be virtually zero in the dark in P-sufficient cells. This shift in intracellular inorganic phosphate concentrations probably reflects changes in the balance between organic phosphates such as ATP and GTP and free phosphate inside the cell in the light and dark. Polarization studies Poolman et al. (1987) suggested that uptake of phosphate in Streptococcus was electroneutral, with a variable number of protons being cotransported with phosphate, depending on the valency of the phosphate being transported. The data shown in Table 4 do not however, point to electroneutral uptake mechanisms. An unexpected observation was the difference in electrogenic behaviour of cells grown in the two media. Cells grown in ASS, which were not P-limited, have an electrical response similar to that previously noted for nitrategrown cells (Ritchie et al., 1997). At pHo 10, where virtually all phosphate is present as HPO42− (Table 3B), added phosphate depolarized the membrane potential indicating a net uptake of cations, not anions. At pHo 7.5, where substantial H2PO4− is present (Table 3A), added phosphate hyperpolarized the membrane potential. P-limited cells (NOXAS medium) showed strong hyperpolarization when offered phosphate at both pHo 7.5 and pHo 10 but less so at pHo 10. There was no depolarization of the membrane potential when P-sufficient cells were offered phosphate at alkaline pHo. This difference in electrical behaviour implies that there are two P-uptake transporters in Synechococcus. These results also indicate that some other ion is involved in phosphate uptake. Valiente & Avendano (1993) and Avendano & Valiente (1994) reported Na+-dependent uptake of phosphate by Synechococcus: similar results were found by Ritchie et al. (1997). Ca2+ has been reported to have an allosteric effect on phosphate transport in Synechococcus (Rigby et al., 1980). On the contrary, we found that the presence or absence of Ca2+ had no measurable effect but our previous work had shown that there was an absolute requirement for Mg2+ (Ritchie et al., 1997). Phosphate requirements of Synechococcus Ammonia-N grown Synechococcus cells that were not phosphate-limited (ASS-grown cells) differed from Punlimited nitrate-grown cells (Ritchie et al., 1997) only in minor respects. They were capable of taking up phosphate at much higher rates (approx. 25 nmol m−2 s−1, Figs 1 and 3) for short lengths of time but these rapid bursts of net phosphate uptake rapidly decreased to the rates required to support sustained growth (< 1 nmol m−2 s−1). When phosphate is freely available, uptake is essentially entirely light-dependent and geared to assimilate any phosphate present and rapidly store it as an insoluble compound, probably primarily as polyphosphate. This store can support several generations of growth. Phosphate-limited Synechococcus cells differ from phosphatesufficient cells in the mechanism used for phosphate uptake. Although the steady-state uptake rate of phosphate by these cells is quite low (approx. 0.1 nmol m−2 s−1), phosphatelimited Synechococcus cells are capable of opportunistically taking up phosphate at very high rates (apptox. 30 nmol m−2 s−1; Fig. 2). In phosphate-sufficient cells, phosphate uptake is, for practical purposes, restricted to illuminated conditions: uptake in the dark is so slow that it is difficult to measure. However, phosphate-limited cells are capable of rapidly taking up phosphate in both light and dark. In the light, phosphate-uptake by phosphate-limited cells is so rapid that it is comparable to net bicarbonate fluxes (Ritchie et al., 1996) www.newphytologist.com © New Phytologist (2001) 152: 189–201 Research and about 60 times the net uptake rate required for sustaining growth. Mechanism of phosphate uptake The Km for uptake of phosphate by P-sufficient cells is similar in nitrate-N (Ritchie et al., 1997) and ammonia-N grown cells and energetically is consistent with an ATP-driven primary transport system. Hydrolysis of ATP produces a driving force of approx. −50 kJ mol−1 (Reid & Walker, 1983; Muchl & Peschek, 1984). The phosphate uptake mechanism would need to have a stoichiometry of one phosphate taken up per ATP to be thermodynamically possible under all the conditions tested in the present study. The stalling potential of the phosphate uptake mechanism is about −40 kJ mol−1, representing a thermodynamic efficiency of about 80%. The genetic database shows that Synechocystis cells have periplasmic phosphate binding proteins (sll0680 and slr1247; Kaneko et al., 1996a,b) that would be expected to play a role in rapid opportunistic uptake of phosphate. Unfortunately, the complete genome of Synechococcus is not yet available but it is known to have a high degree of similarity to that of Synechocystis. The abundance of these proteins would change the Vmax of uptake on a cellular surface area basis without changing the Km and might account for changes in the Vmax for P-sufficient cells grown on ammonia-N and nitrate-N as fixed nitrogen sources. Using the Nernst equation, it can be shown that a 1 PO4 per ATP pump could not decrease external phosphate concentrations to much below 1 mmol m−3. Freshly made up P-free media had an inorganic phosphate concentration well below 0.1 mmol m−3. When cells were incubated in nominally Pfree media for 1–3 h it was found that they lost phosphate to the medium until a steady-state balance between uptake and loss was reached. Extracellular phosphate was approx. 1 mmol m−3 under such conditions. Such an equilibrium is sometimes termed a phosphate compensation point (Fernandez & Garcia-Sanchez, 1994). Phosphate compensation points of approx. 1 mmol m−3 have also been found in the marine red alga, Porphyra (Fernandez & Garcia-Sanchez, 1994). The electrochemical gradient for phosphate under such conditions is approx. 50 kJ mol−1, very near the stalling point of an ATPdriven pump with a stoichiometry of one ATP per phosphate ion taken up. The thermodynamic picture of Synechococcus cells grown under phosphate-limited conditions (NOXAS medium) is essentially similar to that of phosphate-sufficient cells (Ritchie, 1991; Ritchie et al., 1997). However, the apparent Km of the uptake mechanism, its functionality in the dark, and its different electrical behaviour strongly suggest that a different transporter might be involved. Since a pmf-driven (see Table 2) or Na+mf-driven mechanism are both unlikely (Ritchie, 1991; Ritchie, 1998), a primary active transporter driven by ATP appears to be the only plausible uptake mechanism. © New Phytologist (2001) 152: 189–201 www.newphytologist.com P-limited cells (NOXAS) had a much lower Km than that found in phosphate-sufficient cells (ASS), both in this study and in our previous study (Ritchie et al., 1997). Nevertheless, this transporter appears to be another ATP-driven primary transport system. The phosphate uptake mechanism would still need to have a stoichiometry of one phosphate taken up per ATP to be thermodynamically possible. The stalling potential of the phosphate uptake mechanism is approx. −44 kJ mol−1, equivalent to a thermodynamic efficiency of about 90%. Application of the Nernst equation shows that it would not be thermodynamically possible for the P-uptake mechanism of ASS or NOXAS cells to decrease external phosphate concentrations to the nanomolar (µmol m−3) concentrations claimed by Falkner et al. (1984, 1989, 1995) because a driving force greater than that derived from ATP hydrolysis would be required. It is however, worth noting that the strain of Synechococcus used by the Faulkner group (Falkner et al., 1980, 1984) was obtained from oligotrophic waters and may have a phosphate sequestration mechanism quite different from those described in the present study. Assuming that one ATP is used to take up one phosphate, the power consumption for net uptake of phosphate required to sustain growth would be 423 ± 41 pmol m−2 s−1 × 50 kJ mol−1 = 0.021 ± 0.002 mW m−2. For P-limited cells the power consumption is much lower (approx. 0.005 mW m−2) because of the lower P-uptake rate. These values are very low compared to the power demands of uptake of inorganic carbon (Ritchie et al., 1996). However, the power consumption of short bursts of phosphate uptake of 30 nmol m−2 s−1 would be approx. 1.5 mW m−2, corresponding to approx. one half of the demands of net photosynthesis (Ritchie, 1992a; Ritchie et al., 1996). Sustained uptake of phosphate of 10 nmol m−2 s−1 in the dark would be expected to consume much of the energy available from respiration so the effects of such a power demand should be readily observable in oxygen electrode experiments. To sustain very rapid uptake of phosphate, phosphate-limited cells must be capable of rapidly converting into polyphosphate any phosphate that suddenly becomes available. Thus uptake rates based on time scales of several minutes or more measure synthesis of the polyphosphate storage product rather than the transport step across the plasmalemma. Genetic database The thermodynamic evidence points to both a high and a low affinity ATP-driven phosphate pump, each of which has a stoichiometry of 1PO4/ATP. This conclusion is consistent with what is known about phosphate transport genes in cyanobacteria. The complete sequence for one cyanobacterium, Synechocystis PCC 6803 is known (CYANOBASE, http://www.kazusa.or.ip/cyanobase or http://zearth.kazusa.or.jp:8080/cyano/index.html; Kaneko et al., 199 200 Research 1996a,b). Two phosphate transporters (both made up of a periplasmic protein and several other gene products) have been identified as primary ABC-type ATP-driven pumps (sll680, sll681, sll682, sll683, sll684 and slr1247, slr1248, slr1249, slr1250). Both transporters have only one ATPbinding site, which is consistent with a stoichiometry of one PO4 taken up per ATP. The thermodynamics of such pumps place clear limits on how efficiently the cells can scavenge phosphate from the environment. Using the CYANOBASE database, the partial protein sequence (VYAPGTDSGTY DYFNEAILNK ) of the HAPBP (High Affinity Phosphate Binding Protein, approx. 31 kDa) found by Wagner et al. (1994) turns out to have a 79% similarity to the SphX gene (sll0679) of Synechocystis, which is known to be a regulon protein for phosphate transport. Wagner et al. (1994) found the phosphate-binding constant of HAPBP to be about 0.3 mmol m−3 (similar to the Km found in P-limited cells in the present study). It can therefore be concluded that the cluster of genes sll679 to sll684 (SphX, pstA, pstC, pstB, pstB) are likely to represent the transport system described in the present paper. Wagner et al. (1994) identified a second protein (approx. 45 kDa), which was manifested under phosphate limitation, but did not characterize it. We therefore conclude that Synechococcus has a classical high affinity/low affinity transport system for phosphate. The high affinity system (corresponding to genes sll679 to sll684 in Synechocystis) has a Km of approx. 0.3 mmol m−3 and is capable of operating at an appreciable rate in the dark whilst the lower-affinity system (genes slr1247 to slr1250 in Synechocystis) has a Km of approx. 2 mmol m−3 and appears to operate only in the light. Both pumps seem to be driven by ATP. Acknowledgements Dr R. J. Ritchie held an ARC Postdoctoral Research Fellowship during part of this project. Dr Peter Hawkins (Australian Water Technology) kindly provided us with representative analyses of secondary-treated sewage effluent. 32P-H PO was purchased from the Australian Institute of 3 4 Nuclear Science and Engineering (AINSE). Dr Donelle Trautman was supported in part from an ARC Institutional Grant awarded to Dr R. J. Ritchie and Prof A. W. D. Larkum. References Allen MM. 1968. Simple conditions for growth of unicellular blue-green algae. Journal of Phycology 4: 1–3. 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