Arch. Microbiol. 102, 23--28 (1975) 9 by Springer-Verlag 1975
Phosphate Utilization and Alkaline Phosphatase Activity in Anacystis nidulans (Synechococcus) M. J. A. IHLENFELDT and JANE GIBSON Section of Biochemistry, Molecular and Cell Biology Division of Biological Sciences, Cornell University, Ithaca, New York 14 853 Received July 10, 1974
Abstract. Anaeystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30~C. Such cultures had a normal pigment composition and alkaline phosphatase was detectable at low specific activities only. The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 txM (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this
period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate. Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess. Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.
Key words: Anacystis nidulans -- Phosphate Limitation -- Photosynthetic Pigments -- Alkaline Phosphatase.
Alkaline phosphatase expression varies with growth conditions in a number of pro- and eukaryotic cells (Torriani, 1960; Fitzgerald, 1969; K o y a m a and Ono, 1972). Extensive investigations which have been carried out primarily with Eseheriehia eoli have established that enzyme synthesis is repressed when inorganic phosphate is present in the growth medium (Torriani, 1960) and that a complex genetic system is involved in this repression (Echols et aL, 1961). The relationship between enzyme synthesis and phosphate availability has been utilized as an index of phosphate nutrition in aquatic algae (Fitzgerald, 1969). The activity of some enzyme systems involved in carbon metabolism of blue-green algae has been found to be little affected by the composition of the growth medium (Pearce and Carr, 1969; Pelroy et al., 1972) suggesting that phenomena of induction and repression play a smaller part in these organisms than in many heterotrophic prokaryots. However, alkaline phosphatase activity of the filamentous Anabaenaflos-aquae has been shown to vary with medium phosphate composition when grown in continuous culture (Bone, 197/), as well as in batch culture (Healey,/973). In the investigation reported here, derepression of alkaline phosphatase, accompanied by marked changes in photo-
Abbreviations Used. pNP = pnitrophenol; pNPP = pnitrophenylphosphate.
synthetic pigment content of cells ofAnacystics nidulans (Synechoeoeeus) was observed when cultures entered a state of phosphate limitation. The alkaline phosphatase from Anacystis resembled that from other sources in some, but not all, of its properties.
Materials and Methods The strain of Anaeystis nidulans, medium employed, and general culture conditions have been described (Ihlenfeldt and Gibson, 1975). Growth experiments were carried out in acid-cleaned pyrex test tubes or square bottles of 130 ml capacity, which were bubbled continuously with a stream of 5 ~ CO2 in air. Larger cultures for enzyme extraction were grown in Roux bottles, bubbled in the same way. Low phosphate medium containing 0.1 mM K2HPO4 allowed growth to a cell protein concentration of approximately 100 ~tg per ml before changes in cell composition became apparent, and cultures could be transferred repeatedly in this medium. For phosphate starvation a 1 : 10 dilution was made from an exponentially growing culture in this medium into fresh medium prepared without added phosphate; such medium was found to contain between 5 - 8 ~M phosphate by chemical determination. Growth was followed as scattering at 750 nm in a Zeiss PMQ II spectrophotometer, and by protein determination as previously described (Ihlenfeldt and Gibson, 1975). Analytical Methods Pigment Content. Culture samples containing 0.5-0.8 mg cell protein were centrifuged, and the pellets washed twice
24 with buffer-free medium. Pigments were extracted into 3 ml ice-cold acetone : methanol (7:2, v/v; Cohen-Bazire et at., 1957), and chlorophyll calculated from the absorbance at 663 nm, using an extinction coefficient of 82 mM -1 • cm -1. Changes in chlorophyll : carotenoid ratio were detected by determining the absorbance at 480 as well as 663 nm. The acetone : methanol extracted cell pellet was used for protein determinations. Phycocyanin was estimated in sonicated preparations in the following way. A sample containing 0.5 mg cell protein was harvested and washed as above, and suspended in 1 ml 20 mM K phosphate buffer, pH 7.0. The suspension was sonicated for a total of 2 rain at 5~ and centrifuged. The pellet was washed once with 1 ml phosphate buffer, and the absorption spectrum of the combined supernatants scanned with a Cary model 14 recording spectrophotometer. Phycocyanin was determined from the extinction at 618 nm, corrected for the chlorophyll absorbance at this wavelength (Myers and Kratz, 1955), using a specific absorption coefficient of 7.9. The pellet of unbroken cells was extracted with acetone:methanol as above, and a correction factor for cell breakage obtained by comparing the chlorophyll content of the initial unsonicated suspension with that remaining in this residue. Cell breakage was usually in the range of 90--95 ~.
Inorganic Phosphate. This was measured using the procedure described by Dryer et al. (1957). Determination of Enzyme Activity. Samples containing 0.3 to 1 mg cell protein were harvested by centrifugation and suspended in 2 ml 0.5 M Tris- HC1 buffer, pH 8.5. One drop of toluene was added per tube and the samples were equilibrated at 37~ for 15 rain in a water bath. The reaction was started by the addition of 0.1 ml pNPP (final concentration 5.2 raM) and terminated by the addition of 0.1 m l l M K2HPO4, after exactly 5 rain. Samples were centrifuged and the amount of pNP !iberated was measured by the absorbance changes at 400 nm against a blank containing buffer, substrate and phosphate. Preliminary experiments showed that the rate of the reaction was linear for at least 15 rain under the assay conditions employed. The amount of pNP liberated was calculated from a standard curve prepared by dissolving known quantities of pNP in Tris 9 HC1 buffer pH 8.5, 0.5 M; K2HPO4, 0.05 M. Activities measured in whole cells with or without toluene treatment, or in extracts prepared with a French pressure cell agreed within 10~. Relative hydrolysis rates with different phosphate esters were measured at 30~C in 4 ml reaction mixtures containing final concentrations of 5 mM ester, 0.5 M Tris 9H C , pH 8.5 and an appropriate quantity of extracted enzyme (see below). Samples of 1 ml were withdrawn at intervals and mixed with 1 ml 10 ~o trichloracetic acid. Inorganic phosphate released was measured by the procedure of Dryer et al. (1957) as above, except that the final absorbances in pNPP hydrolysis experiments were measured at 770 nm. Inhibition of pNPP hydrolysis by inorganic phosphate, arsenate and ZnCI~ was measured by continuous recording of absorbance changes at 400 nm in a Cary model 14 recording spectrophotometer. Extraction of Alkaline Phosphatase. Suspensions harvested from cultures whose growth had been restricted by phosphate limitation for 20--30 hrs were washed twice in fresh phosphate-free medium, and suspended in 0.25 M sucrose to a concentration of 5 mg cell protein per ml. Lysozyme was added (8 mg per ml) and the suspension incubated at 30~ for between 30 and 90 min, until microscopic examination showed that more than half the cells had changed in shape
Arch. Microbiol., Vol. 102, No. 1 (1975) and refractility. At this stage, a somewhat variable proportion of between 30 and 80 ~ of the total alkaline phosphatase had been released into the colorless supernatant obtained after centrifugation. After dialysis against 20 mM Tris. HC1. pH 7.5, the enzyme was concentrated by adding solid (NH4)2SO4 to 70 ~ saturation. The precipitate was collected and freed of (NH4)2SO4 by dialysis against the same buffer. In some preparations a Sephadex G-75 column (2 • 25 cm) equilibrated with 20 mM Tris 9 HC1, pH 7.5 was used; the alkaline phosphatase emerged with the void volume. Preparations desalted with Sephadex were used for measurements of relative hydrolysis rates with phosphate esters and the inhibitor studies.
Results Growth Experiments Although cultures cou~"-b~, maintained through an indefinite number of transfer's'-in medium containing 0.1 mM phosphate, transfers to medium without added phosphate continued to grow with a normal doubling time for only two to three generations. At the end of this time the culture was yellowish green instead of the normal blue green in color, and the external phosphate concentration had fallen to below 4 [xM, the limit of accurate determination by the method used. Growth as measured by protein concentration slowed dramatically, although OD continued to increase at less than onetwentieth the rate of a normal culture, for at least 40 hrs after the onset of phosphate limitation. Addition of phosphate at any stage during this period resulted in a return to the normal color of the culture and subsequent resumption of growth (Fig. 1) after a lag whose length depended on the duration of phosphate starvation. Attempts were made to correlate the duration of the lag with viability, but were abandoned because of the variable plating efficiency of control cultures. Investigation of pigment content of the cells showed that specific phycocyanin content was sharply reduced during phosphate starvation, while chlorophyll was relatively stable and carotenoid increased, thus accounting for the obvious color change (Table 1). Preferential synthesis of phycocyanin was required before growth could be resumed at the normal rate (Fig. 1).
Alkaline Phosphatase Activity Although alkaline phosphatase activity was detectable in cultures grown in the 0.1 mM phosphate medium, the specific activity was low, and usually about 10 nmoles • rain -1 • mg protein-L Specific activity increased at the start of the period of growth restriction, and continued to rise slowly for at least 20 hrs, reaching final specific activities of 100--150 nmoles • rain -1 • protein -1. Addition of 1 mM phosphate to such cultures almost completely halted synthesis of the
M. J. A. Ihlenfeldt and J. Gibson" Alkaline Phosphatase in Anacystis nidulans
25
Table 1. Cell composition of normal and phosphate deficient Anacystis Normal
7
Phosphate deficient ~
6 b.
O Chlorophyll a (mg x mg protein -x)
0.030
0.033
Phycocyanin (mg • mg protein-*)
0.250
0.180
7 4 .E E x Z
Chlorophyll: carotenoid (peak ratios 663/480)
2
0.88
0.47 c
Protein (mg • mg dry weight-i)
0.48
9
0.42
; Determined 18 hrs after phosphate became growth limiting.
,
o
/ o
7
o/ o
o/o _o-o -----7o ~--------- ~ " ~
~-2 _
9
o*
o.~o
Table 2. Growth rate and alkaline phosphatase activity of cultures using different phosphate sources
r
or,
1.1
Phosphate source (1 mM)
Doubling time Specific (hrs) activity ~
K2HPO4 c~-glycerophosphate fl-glycerophosphate 3-phosphoglycerate 2,3-diphosphoglycerate Glucose 6-phosphate Fructose 6-phosphate None
5.1 7.2 7.5 5.6 5.7 6.3 5.7 --
0 2
b.
-o r~
a
O
~-9 0 9
9
0.1 o
20 TIME
z;O
l;
Hours
~
0.5
-.
1'0 :
Fig. 2. Specific activity of alkaline phosphatase following transfer to medium without added phosphate. At the times indicated by arrows, samples of the culture were withdrawn, supplemented with 1 mM phosphate, and then incubated and sampled in parallel with the main culture
2
o
TIME
60
: Hours
Fig. i. Growth and pigment composition after transfer to medium without added phosphate. Cell density (open symbols) measured as OD750 and phycocyanin/chlorophyll a (filled symbols) measured as OD6zo/OD677. At the times indicated by arrows, portions of the culture were withdrawn, supplemented with 1 mM phosphate, and then incubated and sampled in parallel with the main culture
enzyme, and the specific activity of the cells fell as growth resumed (Fig. 2). A number of phosphate esters could be utilized for growth in place of inorganic phosphate, although the growth rate was lower, and the specific activity of alkaline phosphatase in such cultures was intermediate between that of phosphate saturated and phosphate limited cultures (Table 2). A medium containing 2 mM 3-phosphoglycerate was inoculated from a culture grown on the same phosphate source and allowed to grow from a concentration of 7 to 142 lxg protein x m1-1 in the presence of 35 ~xM a2po4a-. At this time, the cells were centrifuged from the bulk of the culture, and the remainder diluted with the cell-free supernatant and reincubated. A sample taken for radioactivity measure-
9.6 25 39 28 30 36 18 110
Cultures were harvested after two 1:10 transfers in the phosphate source and alkaline phosphatase measured as described in the text. Specific activity: nmoles pNPP hydrolysed x min -I • mg protein ~at 30~
ment showed that 99 ~ of the counts had been taken from the supernantant. However, the diluted culture continued to grow with a 10 hour doubling time for at least 6 doublings with no change in color or appearance of the culture This makes it unlikely that the growth observed in 3-phosphoglycerate could have been due to the presence of a small amount of inorganic phosphate in the ester solution. There was no evidence of diauxic growth when cultures from 0.1 mg phosphate medium were transferred to media containing 30 IxM phosphate and 1 mM glucose 6-phosphate or 3-phosphoglycerate. Properties of Alkaline Phosphatase The enzyme had a broad pH optimum over the range 8--9.5. While the determinations were not carried out
26
Arch. Microbiol., Vol. 102, No. 1 (1975) ]
gA~%3
IJ 40l/
. .J. .
.
Po4 -
.
4
.
8
1 : M 3
I
Fig. 3. Inhibition of alkaline phosphatase by arsenate and phosphate. Double reciprocal plot relating activity (arbitrary units) of extracted alkaline phosphatase in 5 mM pNPP to inhibitor concentration Table 3. Relative rates of phosphate ester hydrolysis by extracted alkaline phosphatase Ester
Relative hydrolysis rate
pNPP AMP c~-glycerophosphate 3-phosphoglycerate Glucose 6-phosphate
1.00 0.414 0.041 0.041 0.027
specifically to measure the pH stability of the enzyme, it is clear that it is also stable to pH at least for the duration of preincubation and assay over the range tested. Half the enzyme activity was lost in 11 min when suspensions of whole cells were incubated at 70°C in 0.5 M Tris • HCI, pH 8.0. The enzyme could be completely inhibited by either inorganic phosphate or arsenate, with half-maximal inhibition at 2.7 and 0.6 mM, respectively (Fig. 3). Surprisingly, Zn 2+, which was added to an assay mixture to determine whether the enzyme had lost metal ion during the extraction procedure, proved to be a potent inhibitor, with a KI of 0.09 raM. Co 2+ and Mn 2+ on the other hand appeared to stimulate enzyme activity, but because of effects on the spontaneous decomposition rate ofpNPP, the phenomena were not clear cut. The relative rates at which a number of phosphate esters were hydrolyzed are given in Table 3. Discussion
The experiments described here show that the alkaline phosphatase activity of Anacystis nidulans depends on environmental conditions in much the same way as that of bacteria and aquatic algae. Activity is low when phosphate needed for growth is supplied as inorganic
phosphate, and increases when 'phosphate supply becomes restricted, or until phosphat e is added back to the medium. Although the enzyme was not extensively purified, it appeared to resemble that from other sources in some of its properties, such as pH optimum, inhibition by inorganic phosphate, and a degree of thermal stability, although this latter property was less marked than is the case with the Escherichia coli enzyme. The relative rates of hydrolysis of phosphate esters are substantially different from those found for E. coli alkaline phosphatase (Garen and kevinthal, 1960). The inhibitory effect of zinc is also unexpected in view of the need for this metal ion in the enzymatically active form of the protein isolated from E. coli (Plocke eta[., 1962). It appears indeed that a number of different metal ions may be involved in the function of the enzyme from different sources, since activity in Anabaena cultures is stimulated by calcium (Healey, 1973). Comparison of specific activity with that given for other organisms under fully expressed conditions is not easy, because of differences in assay conditions. The highest specific activity found in these investigations is slightly higher than that calculated from the data of Fuhs et al. (1972) for Cyclotella nana, or Anabaena flos-aquae grown in continuous culture under phosphate-limited conditions (Bone, 1971) or in batch culture (Healey, 1973), but still only about one tenth that of E. coli (Torriani, 1960). The specific activity of alkaline phosphatase of A. nidulans using an organic phosphate source was considerably below that which could be reached in phosphate starved cultures, but still 3--4 times that of cultures grown in phosphate excess. These results emphasize the limited extent of both repression and derepression in this organism. However, although the enzyme activity of cells growing in adequate phosphate is low, it none the less appeared to be sufficient to allow a smooth transition from an inorganic to an organic phosphate source, as occurred in cultures which contained both a limiting concentration of inorganic phosphate and a higher concentration of ester. The rate at which phosphate must be supplied to support the observed growth rates has been calculated to be about 0.6 nmoles x min -1 × nag protein -1, assuming a ratio of 2:100 (by weight) phosphate : protein in the cells. The activity of alkaline phosphatase in cultures growing with 3-phosphoglycerate as phosphate source averaged 0.9 nmoles × min -1 × mg protein 1 in several experiments, which would thus be adequate to supply the phosphate requirement if hydrolysis occurred at or outside the cell membrane. The strain of A. nidu[ans used here did not contain detectable acid phosphatase. Both the release of enzyme without cell pigment on lysozyme treatment, and the similarity in activity between intact broken ceils suggest strongly that
M. J. A. Ihlenfeldt and J. Gibson: Alkaline Phosphatase in Anacystis nidulans alkaline phosphatase is peripherally located in A. nidulans, as is the case in E. coli (Wetzel et al., 1970). Substantial release of enzyme into the medium on prolonged incubation has been reported for Anabaena (Healey, 1973) but was not observed with the Anacystis strain used here. Continued increases in optical density during phosphate starvation have been observed in E. coli (Torriani, 1960), Anacystis nidulans (Batterton and van Baalen, 1968) and other algae such as Cyclotella (Fuhs et al., 1972) and appear to be due to continued synthesis of storage polysaccharide. Although Batterton and van Baalen observed only minor changes in pigmentation during phosphate experiments, with our strain the loss in phycocyanin and gain in carotenoid resulted in a very marked color change. Since there is only a small change in the specific protein content of cells during phosphate limitation, it appears that it may be only the chromophore of the phycobiliprotein which is degraded, while the protein remains intact. It would be of interest to follow the fate of the apoprotein during these pigment changes. Although measurements of photosynthetic capacity were not carried out during these experiments, Jones and Myers (1964) have shown that phycocyanin-absorbed light is utilized mainly by photosystem II in normal cultures. As is apparent from Fig. 1, growth was not resumed at a normal rate until the cellular phycocyanin had been restored; adequate amounts of this pigment may therefore be necessary for proper balance between the two photosystems. Similar phycocyanin changes were observed by Allen and Smith (1969) following nitrogen starvation of Anacystis, which resulted in a chlorotic change in color following destruction of phycocyanin with, however, little carotenoid change. Changes in the specific content of phycocyanin thus appear to be a sensitive indicator of starvation conditions in Anacystis; we have also observed a relative loss of this pigment during COs limitation, as described by Eley (1971). The possibility of assessing the state of phosphate nutrition of natural populations of algae by the degree of expression of alkaline phosphatase has been investigated and discussed (Fitzgerald, 1969; Healey, 1973). Many general properties of the enzyme, including its prompt control by the availability of phosphate and its stability with respect to temperature and pH, are similar in the strain of Anacystis used here and in other bacteria and algae which have been investigated, suggesting that its activity would indeed give a reasonably accurate picture of the phosphate nutrition of laboratory cultures. However, the fact that the specific activity attainable is much lower than that of other organisms such as E. coli could make interpretation in mixed populations difficult, and there is also the possibility
27
that some strains may lack the enzyme. Kuenzler and Perras (1965) in a broad investigation of a number of marine algae, were not able to demonstrate alkaline or acid phosphatases in all, and indeed list a strain of the blue-green Synechococcus as phosphatase negative. It would obviously be desirable to investigate a number of different blue-green species, since alkaline phosphatase has been investigated only in Anabaena (Bone, 1971 ; Healey, 1973) and here in Anacystis nidulans to assess how useful alkaline phosphatase activity measurements could be with natural populations.
Acknowledgements. This work was supported in part by grant GB 21420 from the National Science Foundation. References Alien, M. M., Smith, A. J. : Nitrogen chlorosis in blue-green algae. Arch. Mikrobiol. 69, 114--120 (1969) Batterton,J. C., van Baalen, C. : Phosphorus deficiency and phosphate uptake in the blue green alga Anacystis nidulans. Canad. J. Microbiol. 14, 341-348 (1968) Bone, D. H. : Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture. Arch. Mikrobiol. 80, 147--153 (1971) Cohen-Bazire, G., Sistrom,W.R., Stanier, R. Y. : Kinetic studies of pigment systhesis by non-sulfuLpurple bacteria. J. cell. comp. Physiol. 49, 25--68 (1957) Dryer, R.L., Tammes, A.R., Routh,J.l.: The determination of phosphorus and phosphatase with N-phenylp-phenylenediamine. J. biol. Chem. 225, 177--183 (1957) Echols, H., Garen,A., Garen, S., Torriani,A.: Genetic control of repression of alkaline phosphat e in E. coll. J. molec. Biol. 3, 425--438 (1961) Eley, J.H.: The effect of carbon dioxide concentration on pigmentation in the blue-green alga Anacystis nidulans. Plant Cell Physiol. 12, 311--316 (1971) Fitzgerald, G.P.: Field and laboratory evaluations of bioassays for nitrogen and phosphorus with algae and aquatic weeds. Limnol. Oceanogr. 14, 206--212 (1969) Fuhs, G.W., Demmerle, S. D., Canelli, E., Chen, M. : Characterization of phosphorus limited plankton algae (with reflections on the limiting nutrient concept). In: G. E. Likens, ed. : Nutrients and eutrophication : the limiting nutrient controversy. American Soc. Limnol. Oceanogr. Special Symp., vol. 1, pp. 113 -- 133. Lawrence, Kansas: Allen Press 1972 Garen,A., Levinthal, C.: A fine structure genetic and chemical study of the enzyme alkaline phosphatase of E. coll. Purification and characterization of the alkaline phosphatase. Biochim. biophys. Acta (Amst.) 38, 470--483 (1960) Healey, F.P.: Characteristics of phosphorus deficiency in Anabaena. J. Phycol. 9, 383 394 (1973) Ihlenfeldt, M. J.A., Gibson, J. : COs fixation and its regulation in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102, 13--21 (1975) Jones, L. W., Myers, J. : Enhancement in the blue-green alga Anacystis nidulans. Plant Physiol. 39, 938-946 (1964)
28 Koyama, H., Ono, T.: Further studies on the induction of alkaline phosphatase by 5-bromodeoxyuridine in a hybrid line between mouse and Chinese hamster in culture. Biochim. biophys. Acta (Amst.) 264, 497-507 (1972) Kuenzler, E.J., Perras, J. P. : Phosphatases of marine algae. Biol. Bull. 128, 271--284 (1965) Myers, J., Kratz,W. A. : Relations between pigment content and photosynthetic characteristics in a blue-green alga. J. gen. Physiol. 39, 11--22 (1955) Pearce, J., Carr, N.G.: The incorporation and metabolism of glucose by Anabaena variabilis. J. gen. Microbiol. 54, 451 --462 (1969)
Arch. Microbiol., Vol. 102, No. 1 (1975) Pelroy, R.A., Rippka, R., Stanier, R.Y.: Metabolism of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87, 303--322 (1972) Plocke, D.J., Levinthal, C., Vallee, B.L.: Alkaline phosphatase of E. coli: a zinc mettalloenzyme. Biochemistry 1,373 --378 (1962) Torriani, A. : Influence of inorganic phosphate in the formation of phosphatases by Escheriehia eoli. Biochim. biophys. Acta (Amst.) 38, 460--469 (1960) Wetzel, B.K., Spicer, S.S., Dvorak, H.F., Heppel, L. A. : Cytochemical localization of certain phosphatases in Escherichia eoli. J. Bact. 104, 529--542 (1970)
Dr. Jane Gibson, Section of Biochemistry, Molecular and Cell Biology, Wing Hall, Cornell University Ithaca, New York 14853, U.S.A.
Phosphate Utilization and Alkaline Phosphatase Activity in Anacystis nidulans (Synechococcus) M. J. A. IHLENFELDT and JANE GIBSON Section of Biochemistry, Molecular and Cell Biology Division of Biological Sciences, Cornell University, Ithaca, New York 14 853 Received July 10, 1974
Abstract. Anaeystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30~C. Such cultures had a normal pigment composition and alkaline phosphatase was detectable at low specific activities only. The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 txM (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this
period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate. Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess. Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.
Key words: Anacystis nidulans -- Phosphate Limitation -- Photosynthetic Pigments -- Alkaline Phosphatase.
Alkaline phosphatase expression varies with growth conditions in a number of pro- and eukaryotic cells (Torriani, 1960; Fitzgerald, 1969; K o y a m a and Ono, 1972). Extensive investigations which have been carried out primarily with Eseheriehia eoli have established that enzyme synthesis is repressed when inorganic phosphate is present in the growth medium (Torriani, 1960) and that a complex genetic system is involved in this repression (Echols et aL, 1961). The relationship between enzyme synthesis and phosphate availability has been utilized as an index of phosphate nutrition in aquatic algae (Fitzgerald, 1969). The activity of some enzyme systems involved in carbon metabolism of blue-green algae has been found to be little affected by the composition of the growth medium (Pearce and Carr, 1969; Pelroy et al., 1972) suggesting that phenomena of induction and repression play a smaller part in these organisms than in many heterotrophic prokaryots. However, alkaline phosphatase activity of the filamentous Anabaenaflos-aquae has been shown to vary with medium phosphate composition when grown in continuous culture (Bone, 197/), as well as in batch culture (Healey,/973). In the investigation reported here, derepression of alkaline phosphatase, accompanied by marked changes in photo-
Abbreviations Used. pNP = pnitrophenol; pNPP = pnitrophenylphosphate.
synthetic pigment content of cells ofAnacystics nidulans (Synechoeoeeus) was observed when cultures entered a state of phosphate limitation. The alkaline phosphatase from Anacystis resembled that from other sources in some, but not all, of its properties.
Materials and Methods The strain of Anaeystis nidulans, medium employed, and general culture conditions have been described (Ihlenfeldt and Gibson, 1975). Growth experiments were carried out in acid-cleaned pyrex test tubes or square bottles of 130 ml capacity, which were bubbled continuously with a stream of 5 ~ CO2 in air. Larger cultures for enzyme extraction were grown in Roux bottles, bubbled in the same way. Low phosphate medium containing 0.1 mM K2HPO4 allowed growth to a cell protein concentration of approximately 100 ~tg per ml before changes in cell composition became apparent, and cultures could be transferred repeatedly in this medium. For phosphate starvation a 1 : 10 dilution was made from an exponentially growing culture in this medium into fresh medium prepared without added phosphate; such medium was found to contain between 5 - 8 ~M phosphate by chemical determination. Growth was followed as scattering at 750 nm in a Zeiss PMQ II spectrophotometer, and by protein determination as previously described (Ihlenfeldt and Gibson, 1975). Analytical Methods Pigment Content. Culture samples containing 0.5-0.8 mg cell protein were centrifuged, and the pellets washed twice
24 with buffer-free medium. Pigments were extracted into 3 ml ice-cold acetone : methanol (7:2, v/v; Cohen-Bazire et at., 1957), and chlorophyll calculated from the absorbance at 663 nm, using an extinction coefficient of 82 mM -1 • cm -1. Changes in chlorophyll : carotenoid ratio were detected by determining the absorbance at 480 as well as 663 nm. The acetone : methanol extracted cell pellet was used for protein determinations. Phycocyanin was estimated in sonicated preparations in the following way. A sample containing 0.5 mg cell protein was harvested and washed as above, and suspended in 1 ml 20 mM K phosphate buffer, pH 7.0. The suspension was sonicated for a total of 2 rain at 5~ and centrifuged. The pellet was washed once with 1 ml phosphate buffer, and the absorption spectrum of the combined supernatants scanned with a Cary model 14 recording spectrophotometer. Phycocyanin was determined from the extinction at 618 nm, corrected for the chlorophyll absorbance at this wavelength (Myers and Kratz, 1955), using a specific absorption coefficient of 7.9. The pellet of unbroken cells was extracted with acetone:methanol as above, and a correction factor for cell breakage obtained by comparing the chlorophyll content of the initial unsonicated suspension with that remaining in this residue. Cell breakage was usually in the range of 90--95 ~.
Inorganic Phosphate. This was measured using the procedure described by Dryer et al. (1957). Determination of Enzyme Activity. Samples containing 0.3 to 1 mg cell protein were harvested by centrifugation and suspended in 2 ml 0.5 M Tris- HC1 buffer, pH 8.5. One drop of toluene was added per tube and the samples were equilibrated at 37~ for 15 rain in a water bath. The reaction was started by the addition of 0.1 ml pNPP (final concentration 5.2 raM) and terminated by the addition of 0.1 m l l M K2HPO4, after exactly 5 rain. Samples were centrifuged and the amount of pNP !iberated was measured by the absorbance changes at 400 nm against a blank containing buffer, substrate and phosphate. Preliminary experiments showed that the rate of the reaction was linear for at least 15 rain under the assay conditions employed. The amount of pNP liberated was calculated from a standard curve prepared by dissolving known quantities of pNP in Tris 9 HC1 buffer pH 8.5, 0.5 M; K2HPO4, 0.05 M. Activities measured in whole cells with or without toluene treatment, or in extracts prepared with a French pressure cell agreed within 10~. Relative hydrolysis rates with different phosphate esters were measured at 30~C in 4 ml reaction mixtures containing final concentrations of 5 mM ester, 0.5 M Tris 9H C , pH 8.5 and an appropriate quantity of extracted enzyme (see below). Samples of 1 ml were withdrawn at intervals and mixed with 1 ml 10 ~o trichloracetic acid. Inorganic phosphate released was measured by the procedure of Dryer et al. (1957) as above, except that the final absorbances in pNPP hydrolysis experiments were measured at 770 nm. Inhibition of pNPP hydrolysis by inorganic phosphate, arsenate and ZnCI~ was measured by continuous recording of absorbance changes at 400 nm in a Cary model 14 recording spectrophotometer. Extraction of Alkaline Phosphatase. Suspensions harvested from cultures whose growth had been restricted by phosphate limitation for 20--30 hrs were washed twice in fresh phosphate-free medium, and suspended in 0.25 M sucrose to a concentration of 5 mg cell protein per ml. Lysozyme was added (8 mg per ml) and the suspension incubated at 30~ for between 30 and 90 min, until microscopic examination showed that more than half the cells had changed in shape
Arch. Microbiol., Vol. 102, No. 1 (1975) and refractility. At this stage, a somewhat variable proportion of between 30 and 80 ~ of the total alkaline phosphatase had been released into the colorless supernatant obtained after centrifugation. After dialysis against 20 mM Tris. HC1. pH 7.5, the enzyme was concentrated by adding solid (NH4)2SO4 to 70 ~ saturation. The precipitate was collected and freed of (NH4)2SO4 by dialysis against the same buffer. In some preparations a Sephadex G-75 column (2 • 25 cm) equilibrated with 20 mM Tris 9 HC1, pH 7.5 was used; the alkaline phosphatase emerged with the void volume. Preparations desalted with Sephadex were used for measurements of relative hydrolysis rates with phosphate esters and the inhibitor studies.
Results Growth Experiments Although cultures cou~"-b~, maintained through an indefinite number of transfer's'-in medium containing 0.1 mM phosphate, transfers to medium without added phosphate continued to grow with a normal doubling time for only two to three generations. At the end of this time the culture was yellowish green instead of the normal blue green in color, and the external phosphate concentration had fallen to below 4 [xM, the limit of accurate determination by the method used. Growth as measured by protein concentration slowed dramatically, although OD continued to increase at less than onetwentieth the rate of a normal culture, for at least 40 hrs after the onset of phosphate limitation. Addition of phosphate at any stage during this period resulted in a return to the normal color of the culture and subsequent resumption of growth (Fig. 1) after a lag whose length depended on the duration of phosphate starvation. Attempts were made to correlate the duration of the lag with viability, but were abandoned because of the variable plating efficiency of control cultures. Investigation of pigment content of the cells showed that specific phycocyanin content was sharply reduced during phosphate starvation, while chlorophyll was relatively stable and carotenoid increased, thus accounting for the obvious color change (Table 1). Preferential synthesis of phycocyanin was required before growth could be resumed at the normal rate (Fig. 1).
Alkaline Phosphatase Activity Although alkaline phosphatase activity was detectable in cultures grown in the 0.1 mM phosphate medium, the specific activity was low, and usually about 10 nmoles • rain -1 • mg protein-L Specific activity increased at the start of the period of growth restriction, and continued to rise slowly for at least 20 hrs, reaching final specific activities of 100--150 nmoles • rain -1 • protein -1. Addition of 1 mM phosphate to such cultures almost completely halted synthesis of the
M. J. A. Ihlenfeldt and J. Gibson" Alkaline Phosphatase in Anacystis nidulans
25
Table 1. Cell composition of normal and phosphate deficient Anacystis Normal
7
Phosphate deficient ~
6 b.
O Chlorophyll a (mg x mg protein -x)
0.030
0.033
Phycocyanin (mg • mg protein-*)
0.250
0.180
7 4 .E E x Z
Chlorophyll: carotenoid (peak ratios 663/480)
2
0.88
0.47 c
Protein (mg • mg dry weight-i)
0.48
9
0.42
; Determined 18 hrs after phosphate became growth limiting.
,
o
/ o
7
o/ o
o/o _o-o -----7o ~--------- ~ " ~
~-2 _
9
o*
o.~o
Table 2. Growth rate and alkaline phosphatase activity of cultures using different phosphate sources
r
or,
1.1
Phosphate source (1 mM)
Doubling time Specific (hrs) activity ~
K2HPO4 c~-glycerophosphate fl-glycerophosphate 3-phosphoglycerate 2,3-diphosphoglycerate Glucose 6-phosphate Fructose 6-phosphate None
5.1 7.2 7.5 5.6 5.7 6.3 5.7 --
0 2
b.
-o r~
a
O
~-9 0 9
9
0.1 o
20 TIME
z;O
l;
Hours
~
0.5
-.
1'0 :
Fig. 2. Specific activity of alkaline phosphatase following transfer to medium without added phosphate. At the times indicated by arrows, samples of the culture were withdrawn, supplemented with 1 mM phosphate, and then incubated and sampled in parallel with the main culture
2
o
TIME
60
: Hours
Fig. i. Growth and pigment composition after transfer to medium without added phosphate. Cell density (open symbols) measured as OD750 and phycocyanin/chlorophyll a (filled symbols) measured as OD6zo/OD677. At the times indicated by arrows, portions of the culture were withdrawn, supplemented with 1 mM phosphate, and then incubated and sampled in parallel with the main culture
enzyme, and the specific activity of the cells fell as growth resumed (Fig. 2). A number of phosphate esters could be utilized for growth in place of inorganic phosphate, although the growth rate was lower, and the specific activity of alkaline phosphatase in such cultures was intermediate between that of phosphate saturated and phosphate limited cultures (Table 2). A medium containing 2 mM 3-phosphoglycerate was inoculated from a culture grown on the same phosphate source and allowed to grow from a concentration of 7 to 142 lxg protein x m1-1 in the presence of 35 ~xM a2po4a-. At this time, the cells were centrifuged from the bulk of the culture, and the remainder diluted with the cell-free supernatant and reincubated. A sample taken for radioactivity measure-
9.6 25 39 28 30 36 18 110
Cultures were harvested after two 1:10 transfers in the phosphate source and alkaline phosphatase measured as described in the text. Specific activity: nmoles pNPP hydrolysed x min -I • mg protein ~at 30~
ment showed that 99 ~ of the counts had been taken from the supernantant. However, the diluted culture continued to grow with a 10 hour doubling time for at least 6 doublings with no change in color or appearance of the culture This makes it unlikely that the growth observed in 3-phosphoglycerate could have been due to the presence of a small amount of inorganic phosphate in the ester solution. There was no evidence of diauxic growth when cultures from 0.1 mg phosphate medium were transferred to media containing 30 IxM phosphate and 1 mM glucose 6-phosphate or 3-phosphoglycerate. Properties of Alkaline Phosphatase The enzyme had a broad pH optimum over the range 8--9.5. While the determinations were not carried out
26
Arch. Microbiol., Vol. 102, No. 1 (1975) ]
gA~%3
IJ 40l/
. .J. .
.
Po4 -
.
4
.
8
1 : M 3
I
Fig. 3. Inhibition of alkaline phosphatase by arsenate and phosphate. Double reciprocal plot relating activity (arbitrary units) of extracted alkaline phosphatase in 5 mM pNPP to inhibitor concentration Table 3. Relative rates of phosphate ester hydrolysis by extracted alkaline phosphatase Ester
Relative hydrolysis rate
pNPP AMP c~-glycerophosphate 3-phosphoglycerate Glucose 6-phosphate
1.00 0.414 0.041 0.041 0.027
specifically to measure the pH stability of the enzyme, it is clear that it is also stable to pH at least for the duration of preincubation and assay over the range tested. Half the enzyme activity was lost in 11 min when suspensions of whole cells were incubated at 70°C in 0.5 M Tris • HCI, pH 8.0. The enzyme could be completely inhibited by either inorganic phosphate or arsenate, with half-maximal inhibition at 2.7 and 0.6 mM, respectively (Fig. 3). Surprisingly, Zn 2+, which was added to an assay mixture to determine whether the enzyme had lost metal ion during the extraction procedure, proved to be a potent inhibitor, with a KI of 0.09 raM. Co 2+ and Mn 2+ on the other hand appeared to stimulate enzyme activity, but because of effects on the spontaneous decomposition rate ofpNPP, the phenomena were not clear cut. The relative rates at which a number of phosphate esters were hydrolyzed are given in Table 3. Discussion
The experiments described here show that the alkaline phosphatase activity of Anacystis nidulans depends on environmental conditions in much the same way as that of bacteria and aquatic algae. Activity is low when phosphate needed for growth is supplied as inorganic
phosphate, and increases when 'phosphate supply becomes restricted, or until phosphat e is added back to the medium. Although the enzyme was not extensively purified, it appeared to resemble that from other sources in some of its properties, such as pH optimum, inhibition by inorganic phosphate, and a degree of thermal stability, although this latter property was less marked than is the case with the Escherichia coli enzyme. The relative rates of hydrolysis of phosphate esters are substantially different from those found for E. coli alkaline phosphatase (Garen and kevinthal, 1960). The inhibitory effect of zinc is also unexpected in view of the need for this metal ion in the enzymatically active form of the protein isolated from E. coli (Plocke eta[., 1962). It appears indeed that a number of different metal ions may be involved in the function of the enzyme from different sources, since activity in Anabaena cultures is stimulated by calcium (Healey, 1973). Comparison of specific activity with that given for other organisms under fully expressed conditions is not easy, because of differences in assay conditions. The highest specific activity found in these investigations is slightly higher than that calculated from the data of Fuhs et al. (1972) for Cyclotella nana, or Anabaena flos-aquae grown in continuous culture under phosphate-limited conditions (Bone, 1971) or in batch culture (Healey, 1973), but still only about one tenth that of E. coli (Torriani, 1960). The specific activity of alkaline phosphatase of A. nidulans using an organic phosphate source was considerably below that which could be reached in phosphate starved cultures, but still 3--4 times that of cultures grown in phosphate excess. These results emphasize the limited extent of both repression and derepression in this organism. However, although the enzyme activity of cells growing in adequate phosphate is low, it none the less appeared to be sufficient to allow a smooth transition from an inorganic to an organic phosphate source, as occurred in cultures which contained both a limiting concentration of inorganic phosphate and a higher concentration of ester. The rate at which phosphate must be supplied to support the observed growth rates has been calculated to be about 0.6 nmoles x min -1 × nag protein -1, assuming a ratio of 2:100 (by weight) phosphate : protein in the cells. The activity of alkaline phosphatase in cultures growing with 3-phosphoglycerate as phosphate source averaged 0.9 nmoles × min -1 × mg protein 1 in several experiments, which would thus be adequate to supply the phosphate requirement if hydrolysis occurred at or outside the cell membrane. The strain of A. nidu[ans used here did not contain detectable acid phosphatase. Both the release of enzyme without cell pigment on lysozyme treatment, and the similarity in activity between intact broken ceils suggest strongly that
M. J. A. Ihlenfeldt and J. Gibson: Alkaline Phosphatase in Anacystis nidulans alkaline phosphatase is peripherally located in A. nidulans, as is the case in E. coli (Wetzel et al., 1970). Substantial release of enzyme into the medium on prolonged incubation has been reported for Anabaena (Healey, 1973) but was not observed with the Anacystis strain used here. Continued increases in optical density during phosphate starvation have been observed in E. coli (Torriani, 1960), Anacystis nidulans (Batterton and van Baalen, 1968) and other algae such as Cyclotella (Fuhs et al., 1972) and appear to be due to continued synthesis of storage polysaccharide. Although Batterton and van Baalen observed only minor changes in pigmentation during phosphate experiments, with our strain the loss in phycocyanin and gain in carotenoid resulted in a very marked color change. Since there is only a small change in the specific protein content of cells during phosphate limitation, it appears that it may be only the chromophore of the phycobiliprotein which is degraded, while the protein remains intact. It would be of interest to follow the fate of the apoprotein during these pigment changes. Although measurements of photosynthetic capacity were not carried out during these experiments, Jones and Myers (1964) have shown that phycocyanin-absorbed light is utilized mainly by photosystem II in normal cultures. As is apparent from Fig. 1, growth was not resumed at a normal rate until the cellular phycocyanin had been restored; adequate amounts of this pigment may therefore be necessary for proper balance between the two photosystems. Similar phycocyanin changes were observed by Allen and Smith (1969) following nitrogen starvation of Anacystis, which resulted in a chlorotic change in color following destruction of phycocyanin with, however, little carotenoid change. Changes in the specific content of phycocyanin thus appear to be a sensitive indicator of starvation conditions in Anacystis; we have also observed a relative loss of this pigment during COs limitation, as described by Eley (1971). The possibility of assessing the state of phosphate nutrition of natural populations of algae by the degree of expression of alkaline phosphatase has been investigated and discussed (Fitzgerald, 1969; Healey, 1973). Many general properties of the enzyme, including its prompt control by the availability of phosphate and its stability with respect to temperature and pH, are similar in the strain of Anacystis used here and in other bacteria and algae which have been investigated, suggesting that its activity would indeed give a reasonably accurate picture of the phosphate nutrition of laboratory cultures. However, the fact that the specific activity attainable is much lower than that of other organisms such as E. coli could make interpretation in mixed populations difficult, and there is also the possibility
27
that some strains may lack the enzyme. Kuenzler and Perras (1965) in a broad investigation of a number of marine algae, were not able to demonstrate alkaline or acid phosphatases in all, and indeed list a strain of the blue-green Synechococcus as phosphatase negative. It would obviously be desirable to investigate a number of different blue-green species, since alkaline phosphatase has been investigated only in Anabaena (Bone, 1971 ; Healey, 1973) and here in Anacystis nidulans to assess how useful alkaline phosphatase activity measurements could be with natural populations.
Acknowledgements. This work was supported in part by grant GB 21420 from the National Science Foundation. References Alien, M. M., Smith, A. J. : Nitrogen chlorosis in blue-green algae. Arch. Mikrobiol. 69, 114--120 (1969) Batterton,J. C., van Baalen, C. : Phosphorus deficiency and phosphate uptake in the blue green alga Anacystis nidulans. Canad. J. Microbiol. 14, 341-348 (1968) Bone, D. H. : Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture. Arch. Mikrobiol. 80, 147--153 (1971) Cohen-Bazire, G., Sistrom,W.R., Stanier, R. Y. : Kinetic studies of pigment systhesis by non-sulfuLpurple bacteria. J. cell. comp. Physiol. 49, 25--68 (1957) Dryer, R.L., Tammes, A.R., Routh,J.l.: The determination of phosphorus and phosphatase with N-phenylp-phenylenediamine. J. biol. Chem. 225, 177--183 (1957) Echols, H., Garen,A., Garen, S., Torriani,A.: Genetic control of repression of alkaline phosphat e in E. coll. J. molec. Biol. 3, 425--438 (1961) Eley, J.H.: The effect of carbon dioxide concentration on pigmentation in the blue-green alga Anacystis nidulans. Plant Cell Physiol. 12, 311--316 (1971) Fitzgerald, G.P.: Field and laboratory evaluations of bioassays for nitrogen and phosphorus with algae and aquatic weeds. Limnol. Oceanogr. 14, 206--212 (1969) Fuhs, G.W., Demmerle, S. D., Canelli, E., Chen, M. : Characterization of phosphorus limited plankton algae (with reflections on the limiting nutrient concept). In: G. E. Likens, ed. : Nutrients and eutrophication : the limiting nutrient controversy. American Soc. Limnol. Oceanogr. Special Symp., vol. 1, pp. 113 -- 133. Lawrence, Kansas: Allen Press 1972 Garen,A., Levinthal, C.: A fine structure genetic and chemical study of the enzyme alkaline phosphatase of E. coll. Purification and characterization of the alkaline phosphatase. Biochim. biophys. Acta (Amst.) 38, 470--483 (1960) Healey, F.P.: Characteristics of phosphorus deficiency in Anabaena. J. Phycol. 9, 383 394 (1973) Ihlenfeldt, M. J.A., Gibson, J. : COs fixation and its regulation in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102, 13--21 (1975) Jones, L. W., Myers, J. : Enhancement in the blue-green alga Anacystis nidulans. Plant Physiol. 39, 938-946 (1964)
28 Koyama, H., Ono, T.: Further studies on the induction of alkaline phosphatase by 5-bromodeoxyuridine in a hybrid line between mouse and Chinese hamster in culture. Biochim. biophys. Acta (Amst.) 264, 497-507 (1972) Kuenzler, E.J., Perras, J. P. : Phosphatases of marine algae. Biol. Bull. 128, 271--284 (1965) Myers, J., Kratz,W. A. : Relations between pigment content and photosynthetic characteristics in a blue-green alga. J. gen. Physiol. 39, 11--22 (1955) Pearce, J., Carr, N.G.: The incorporation and metabolism of glucose by Anabaena variabilis. J. gen. Microbiol. 54, 451 --462 (1969)
Arch. Microbiol., Vol. 102, No. 1 (1975) Pelroy, R.A., Rippka, R., Stanier, R.Y.: Metabolism of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87, 303--322 (1972) Plocke, D.J., Levinthal, C., Vallee, B.L.: Alkaline phosphatase of E. coli: a zinc mettalloenzyme. Biochemistry 1,373 --378 (1962) Torriani, A. : Influence of inorganic phosphate in the formation of phosphatases by Escheriehia eoli. Biochim. biophys. Acta (Amst.) 38, 460--469 (1960) Wetzel, B.K., Spicer, S.S., Dvorak, H.F., Heppel, L. A. : Cytochemical localization of certain phosphatases in Escherichia eoli. J. Bact. 104, 529--542 (1970)
Dr. Jane Gibson, Section of Biochemistry, Molecular and Cell Biology, Wing Hall, Cornell University Ithaca, New York 14853, U.S.A.
