Phosphate uptake kinetics and its regulation in N2- fixing cyanobacterium Anabaena oryzae Fritsch under salt stress

Phosphorous (P) starved cells of the cyanobacterium Anabaena oryzae showed higher phosphate uptake rates than P-sufficient cells. The P-uptake obeyed saturation kinetics. The Km value for Pdeficient cells was lower (54.34 μM) than P-sufficient cells (82.64 μM) while Vmax was higher in Pdeficient and lower in P-sufficient cells. Salinity (NaCl) stimulated phosphate uptake significantly in the cyanobacterium which is followed by greater amount of P-accumulation in the form of polyphosphate bodies. Inhibition of P-uptake in P-deficient cells was 45% in dark grown compared to light grown cells. P-uptake was inhibited 52 and 85% in culture treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 10 μM) and carbonyl cyanide m-chloro phenylhydrazone (CCCP; 100 μM), respectively, suggesting that energy for uptake could be derived from oxidative phosphorylation and photophosphorelation.


INTRODUCTION
Phosphorous (P) in municipal, agricultural and industrial effluents is an important contributory factor to eutrofication and its recycling for further utilization is a matter of concern. It is widely accepted that phosphorus is the main nutrient that controls the development of natural populations of cyanobacteria in many terrestrial fresh water and marine environments (Healey, 1982;Mann, 1994;Whitton and Pott, 2000;Singh et al., 2006).
Phosphorus storage in cyanobacteria appears to be much larger than in other species and this capacity was reported to give them a competitive advantage over diatoms and chlorophytes when P was supplied in pulses (Sommer, 1985;Reinertsen et al., 1986;Sakshaug and Olsen, 1986). Phosphate uptake in cyanobacteria follows saturation type kinetics with kinetics parameters (K ½ and V max ) comparable to those of eukaryotic algae (Healey, 1982). The uptake process is energy-dependent (Healey, 1982;Budd and Kerson, 1987;Fernandez Valiente and Avendano, 1993) but its mechanism is unclear.
Phosphate uptake in cyanobacteria also requires speci-fic cations. A stimulation of phosphate uptake by divalent cations Mg 2+ , Ca 2+ and Sr 2+ has been reported in several strains of N 2 -fixing cyanobacteria (Falkner et al., 1980;Budd and Kerson, 1987). The cation requirement for the increased phosphate uptake and alkaline phosphatase activity suggest for their close functional relations and can be interpreted as adaptive responses to phosphorus limitation. Na + stimulation of phosphate uptake has been reported in cyanobacteria (Fernandez et al., 1993;Avendano and Fernandez Valiente, 1994).
A. oryzae is a dominant cyanobacterium strain of ricefield throughout the crop season where it can be used as biofertilizer. The potential of A. oryzae to take up phosphorus by phosphate starved cells was investigated under the influence of light and dark, metabolic inhibitors and salinity.

Organism and growth conditions
The heterocystous cyanobacterium A. oryzae, a local isolates from rice field of Banaras Hindu University agriculture farm was cultivated and maintained in eight fold diluted Allen-Arnon medium (AA/8) with A5 trace element (Allen and Arnon, 1955). The clonal and axenic population of cyanobacteria was raised and their purity was tested following the standard microbiological techniques. The strain was identified using the classical and recent literatures (Desikachary, 1959;Rippka et al., 1979). The cultures were incubated for photoautotrophic growth in a culture cabinet under cool fluorescent illumination (25 µ Em -2 s -1 ) for 14 h a day and at a temperature of 30 ± 2°C. The experiments were performed under diazotrophic growth condition (N2-medium) and repeated 3 to 4 times. The pH of the growth medium was maintained 7.6 with HEPES buffer at a final concentration of 2 mM.

Cellular protein content
The total protein contents of cultures were measured following the methods of Lowry et al. (1951) as modified by Herbert et al. (1971).

Phosphate uptake
Five days old cultures of the cyanobacterium A. oryzae were harvested, washed twice with P-deficient Allen-Arnon medium (AA/8) and suspended in P-sufficient or P-deficient medium for 12 h under standard growth conditions and termed as P-sufficient and Pdeficient cells. The cells were collected, washed thoroughly, resuspended in P-deficient Allen-Arnon medium (AA/8) containing different concentrations of NaCl, and incubated under standard growth conditions. After an hour of preincubation, assay was started by adding K2HPO4 to a final concentration of 400 µM. In another set of experiment NaCl concentration remain constant (8 mM) while K2HPO4 concentration varied 50 -400 µM. At different time intervals, aliquots were withdrawn and filtered to remove the cells. The filtrates were used to determine the phosphate concentration according to Healey (1978). P uptake was calculated from its depletion in the external medium.

Cellular phosphate
Cyanobacterial cells for determination of cellular phosphorus were collected by centrifugation and washed twice with sterile double dis-tilled water. Samples were dried at 85°C, till constant weight, followed by digestion in boiling concentrated HNO3 and H2SO4 (American Public Health Association (APHA), 1995). The phosphorrus of the digest was determined by the ascorbic acid method (APHA, 1995).

Polyphosphate content
The total polyphosphate granules of the cells were extracted following the method of Poindexter and Fay (1983), and analyzed the Pi following the method of Healey (1978).

Phosphate uptake
The rate of P-uptake by P-starved cell increased with increase in phosphate concentrations up to 400 µM in a concentration-dependent manner and further increase in P-concentration in assay medium has no effect on uptake process. Rates of P-uptake determined for P-sufficient cells also showed the similar trend of uptake and the rate did not increase above the 200 µM. P-uptake in A. oryzae follows saturation type kinetics ( Figure 1A). The 2.3 fold increase in P-uptake by P-deficient cells indicates the role of cellular P-level in controlling the process of Puptake.
The Km value was lower (54.34 µMP) in P-deficient than P-sufficient cells (82.64 µMP) while V max value was high (0.11 µmol P mg protein -1 min -1 ) for P-deficient and low (0.06 µmol P mg protein -1 min -1 ) for P-sufficient cells ( Figure 1B). This shows that P-starvation of cells increases the rate of phosphate transport, which is much higher than the rate observed for P-sufficient cells. The P-level of cells under these conditions is attributed to be a factor controlling the phosphate transport function.

Effect of light/dark and metabolic inhibitors on phosphate uptake
The energy-dependence of phosphate uptake process was observed in light and dark and in the presence of different photosynthesis and electron-transport chain inhibitors. The cells were phosphate starved for 24 h and incubated with phosphate (400 µM) in combination with or without inhibitors. The uptake rates were determined after 4 hours of incubation The P-uptake in the presence of light (control condition) was 5.15 µmol P mg protein -1 and in dark, the uptake activity was reduced to about 55% indicating the light energy depend uptake process (Table 1). DCMU (10 µM), in the photosynthetic growth condition inhibited 52% P-uptake. Arsenate, a potent inhibitor of respiration and analogue of phosphate had no effect on uptake at low concentration (25 µM). Even at higher concentrations (50, 100 µM), the percent inhibition of phosphate uptake was relatively less. CCCP (100 µM),  an inhibitor of electron transport chain, severely inhibited P-uptake (85% activity) in cyanobacterium.

Effect of NaCl
The requirement of Na + in phosphate uptake was examined in P-starved Anabaena cells in the presence of 400 µM phosphate supplemented with different concentration of NaCl (0.5 -10 mM). Phosphate uptake was periodically measured with respect to time as usual. In this short term P-uptake experiments, the data presented ( Figure 2a) illustrate the response of phosphate uptake to increasing concentration of Na + . The level of phosphate uptake by Anabaena cells increased with the increase in concentration of Na + upto a saturation level (8 mM), above which no further increase or decrease occurred, atleast in the range of Na + concentrations tested. The results presented here indicate the stimulatory effect of Na + on phosphate uptake. In another experiment, the effect of Na + on P-uptake was also examined in the presence of different concentrations of phosphate (50 -400 µM). As shown in Figure 2b the P-uptake level in the cyanobacterium was  significantly reduced when cells were incubated in the absence of Na + . A two-fold decrease in P-uptake was noted in the absence of Na + . However, the uptake activity was found to be dependent on phosphate concentrations irrespective of the presence or absence of NaCl (8 mM) in the medium and the activity increased with respect to phosphate concentration. The considerably enhanced Puptake in the presence of NaCl indicates the requirement of Na + in the uptake process in A. oryzae.

Effect of salinity on cellular and polyphosphate of Anabaena oryzae
Cyanobacteria, under phosphate sufficient condition accumulate polyphosphate in the form of granules which is comparatively more under diazotrophically grown cells.
The effect of sodium chloride on cellular P-uptake and phosphate accumulation in the form of polyphosphate showed that the cell-P increased with increment in NaCl (0 -10 mM) level. Cellular-P was (3.0 mg P g -1 cell protein) minimum in absence of NaCl and sodium chloride stimulated which was maximum (3.63 mg P g -1 cell protein) in NaCl (8 mM) supplemented medium. Similar trend was also observed in poly P-accumulation in cell.
There seems to exist a positive correlation between poly-P-accumulation and cellular P-content. Poly-P contributed about 10% to cellular P-content and sodium chloride enhanced accumulation up to 26% of the total cell P (Figure 3).

DISCUSSION
Microbes, including cyanobacteria, have evolved the potential to respond to environmental changes by changing themselves. This potential for rapid adaptation is uni-quely associated with the life style of microbes and resides in their genome which carries all the information necessary for organism to confer upon them the ability to respond to changes in their environment. Thus an organism of a given genotype is a very much product of its environment (Tempest and Neigssel, 1978). One of the environmental parameters in natural ecosystems is the frequent depletion of one or more essential nutrients which commonly influence the properties of microbial cells in nature and cultural conditions, and for that microbes, it possess a wide variety of mechanisms for adapting them self both structurally and functionality with the objective to ensure their prolonged survival in the environment.
Phosphate uptake by P-sufficient cells was saturated at 200 µM phosphate concentration while P-deficient cells had a saturation point at 400 µM concentration. The 2-3 fold increase in P-uptake by P-deficient cells over Psufficient cells also indicates the role of cellular P-level in regulating the uptake process. Like other microbial systems, the P-uptake in A. oryzae also follows saturation kinetics. The Km for P-deficient cells was lower than the P-sufficient cells while V max was higher in P-deficient and lower in P-sufficient cells. This provides evidence to suggest that P-starvation of cells increases the phosphate transport rate. This important function was also found to be energy regulated as the activity was greatly reduced in dark or in cells treated with inhibitors of photosynthesis and respiration. The requirement of energy for P-uptake varied under various Pi concentrations in the medium. The very low concentration of available phosphate (Pi) in the soil (Ragothama, 1999) and a high demand for Pi in cell poses a unique problem. A. oryzae adopted strategies to overcome this low Pi concentration problem by increasing Pi affinity through decrease in Km value (NaCl mediated) or energy provided by ATP. It was of interest to note that this process also required Na + for optimal activity which increased with increasing concentration of phosphate. The uptake rate was three fold higher in the presence of NaCl than its absence. The kinetic parameter of P-uptake was also affected by NaCl that is, the presence of NaCl significantly increased V max and decreased Km value, compared to the control. This confirms the previous report on Na + -stimulation of P-uptake in unicellular and filamentous cyanobacteria (Apte and Thomas, 1985;Avendano and Fernandez Valiente, 1994). Energy required for P-uptake can be provided either by ATP or by electro-chemical gradient. The possibility of Na + /phosphate cotransport has been envisaged as has been reported for Na + -dependent nitrate transport in Anacystis nidulans (Rodriguez et al., 1992) and for phosphate uptake in the heterotrophic bacterium Streptococcus faecalis (Rabaste et al., 1991) and in some non-nitrogen fixing cyanobacteria (Avendano and Valiente, 1994).
Evidences have been presented against the involvement of proton motive force in the process of phosphate Singh et al. 2367 uptake (Budd and Kerson, 1987). The possibility that Na + -electrochemical gradient can act as energy source for this process must be however taken into consideration. Illuminated cyanobacteria maintain a downhill Na + electrochemical gradient from outside to inside which can provide energy for secondary active transport (Ritchie, 1992).
Cyanobacteria growing under P-stress evolve several strategies to cope up in the P-deficiency. Mechanisms involved include the synthesis of phosphates, high efficiency for P-uptake and utilization of its stored polyphosphate. The gene(s) involved in the regulation of these process are located on different operons (Bhaya et al., 2000) however, it seems to belong to the same gene family having different regulon under similar physiological condition of P-stress. Another physiological stress created by NaCl acts synergistically to the above mentioned functions which are of great practical significance. The findings are important and can be exploited biotechnologically in the production of APases and removal of Pi through uptake process and its accumulation in the form of poly-P body.