Vol. 139, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1979, p. 639-645 0021-9193/79/08-0639/07$02.00/0
Phosphate-Limited Culture of Azotobacter vinelandii J. C. TSAI, S. L. ALADEGBAMI, AND G. R. VELA* Department of Biological Sciences, North Texas State University, Denton, Texas 76203
Received for publication 16 May 1979
Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly,8-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.
Although the Azotobacteriaceae have been extensively studied for almost 100 years (7, 11, 24, 32) and although it has long been known that phosphorous (phosphate) is a vital element in all living cells, little is known of the effect of phosphate deprivation on the physiology of these bacteria. Although the role of phosphate in the structure and function of other microorganisms has been studied in great detail (9, 20, 23) only a few reports regarding specific effects of phosphate limitation on Azotobacter appear in the literature (3, 4, 13, 19). Of these, the work which describes the loss of cell viability in phosphate-deficient cultures of Azotobacter chroococcum (3, 4, 13) raises provocative and important questions. Dalton and Postgate (3, 4) reported growth inhibition and decreased viability in phosphate. limited cultures and found a correlation between cellular oxygen consumption and nitrogenase function. This led them to the conclusion that phosphate-limited cultures stop growing in the presence of oxygen because ADP is converted to ATP and the resulting increased ratio of ATP to ADP "shuts off" respiration. They further postulated that this allows oxygen to accumulate in the cell, causing the inactivation of the nitrogenase system. Lees and Postgate (13) reiterated this explanation and offered supporting evidence' by showing that phosphate-sufficient cells remove dissolved oxygen from culture fluid, whereas phosphate-deficient cells fail to do so under the same conditions. From these reports, it appears that decreased viability of cells grown in phosphate-deficient media was due to oxidation of nitrogenase, which was a consequence of the phosphorylation of ADP.
This is a report on measurements of the adenylate pools in cells of Azotobacter vinelandii grown in phosphate-deficient media. The experiments were conducted in batch cultures rather than chemostat cultures since the former show better the effect of phosphate limitation during different physiological states of the organism in question. Also, the conditions of the experiments, i.e., vigorous aeration with consequent maximal oxygenation of the culture medium, permitted us to distinguish the effects of phosphate deficiency from those of oxygen toxicity. A recent report (10) seriously questions the idea of oxygen toxicity and, in this respect, is in agreement with some of the data reported here. MATERIALS AND METHODS
Cultures. Stock cultures of A. vinelandii ATCC 12837 were maintained on Burk nitrogen-free agar slants (31) and were streaked onto plates or inoculated into liquid media when needed. Burk medium contained 640 mg of K2HPO4 and 160 mg of KH2PO4 per liter of water. Phosphate-deficient media were prepared with only 6.4 mg of K2HPO4 and 1.6 mg of KH2PO4 per liter of water. Normal or phosphate-deficient encystment media were prepared by replacing glucose with filter-sterilized n-butanol to a final concentration of 0.3%. Comparisons between normal cells and those grown in phosphate-deficient media were made from cultures started with identical inocula from the same seed culture. The pH's of both normal and low-phosphate cultures were periodically monitored and adjusted to 7.3 by adding 0.1 N KOH. All cultures were grown at 30°C, and liquid cultures were incubated with shaking to insure adequate aeration, i.e., a residual oxygen level of more than 1 mg of dissolved 02 per liter. Chemical determinations. The protein content of both control and phosphate-deficient cells was 639
640
TSAI, ALADEGBAMI, AND VELA
measured by the method of Murphy and Kiew (18) and also by the method of Warburg and Christian (30). The two were run simultaneously for comparison. The Warburg-Christian method (30) for nucleic acid assay was used throughout these studies, and the procedure described by Senior et al. (25) was used for all determinations of poly-f,-hydroxybutyric acid (PHB). The method of Bligh and Dyer (1) was used for determination of total cell lipids, and phosphates were measured by the stannous chloride method described by Sletten and Bach (26). Adenylate charge. The procedure suggested by Chapman et al. (2) was used for measuring ATP, ADP, and AMP and also for calculating the energy charge of the adenylate pool. There were, however, two significant departures from the published method: (i) 50 mg of buffered firefly lantern extract (Sigma Chemical Co., St. Louis, Mo.) was reconstituted in 5.0 ml of deionized water, stored at -20°C overnight, and filtered through a glass fiber pad before use; and (ii) a model 2000 integrating photometer (SAI Scientific Co., San Diego, Calif.) was used in all measurements. Reference curves for ATP were linear and reproducible in the range of 10-12 to 10-`5 mol of ATP in the reaction mixture. All other enzymes and substrates required for these assays were obtained from Sigma Chemical Co. Respiratory activities. Oxygen uptake of whole cells was determined at 30°C in a Gilson model KlC Oxygraph with a Clark-type electrode (Gilson Medical Electronics, Inc., Middleton, Wis.).
Encystment studies. Cultures of A. vinelandii were grown to late log phase in phosphate-sufficient and phosphate-deficient media. The cells were washed twice by centrifugation and plated on encystment media containing the same amount of phosphate as the growth medium. Cultures were examined for cyst formation by a previously described staining procedure
J. BACTERIOL.
chosen from preliminary studies because it yielded approximately 60% the number of cells found in control cultures. The growth rate during the early log phase was essentially the same as in control cultures, although the lag phase was slightly longer. Further studies showed that cells grown in phosphate-deficient media contained approximately the same amount of nucleic acid and protein as did cells grown in phosphate-sufficient media (Table 1). Another study (Table 2) showed that the amount of phosphate in the medium had only a minor effect on cell DNA and RNA. These data imply that cells grown in phosphate-deficient media were different from control cells in some respects but not in others. For example, although the medium designated phosphate-deficient contained only 1/100 the amount of phosphate present in the control medium, the amount of phosphate in the cells from the two cultures was essentially the same (Fig. 2). It can be inferred from the data in Fig. 2 that cell division was accomplished by phosphate sharing and that each daughter cell received an adequate allotment of available phosphate. When the total amount fell below a given level, cell division ceased. It appears certain, then, that these were normal cells adapted to a growth environment where the dearth of phosphate affected (determined) the total cell yield of the cultures but had little effect on other aspects of cell growth, such as total protein and nucleic acids. On the other hand, when phosphate-limited
(29).
Electron microscopy. Cells were harvested by centrifugation, washed three times in distilled water, and suspended in 3% glutaraldehyde buffered with 0.1 M sodium cacodylate. They were fixed for 1 h as described by Vela et al. (28), washed again, and then placed in 1% (vol/vol) OS04 in cacodylate buffer at 4°C for 1 h. They were again washed and then dehydrated by passing through 30, 50, 75, 85, 95, and 100% (vol/vol) ethanol solutions before embedding in Epon 812. Thin sections were cut with a Porter-Blum ultramicrotome (Ivan Sorvall, Inc., Norwalk, Conn.) equipped with a glass knife. All sections were stained with uranyl acetate for 45 min and then with tartrate for 30 min and examined with an RCA-EMU-3G electron microscope at initial magnifications of x11,500 to x16,000.
9.5 r
9.0 .5a 'a z
8.5 h 0'
/
8.0 -,1 '
80 70 -E 60 U 50 :L .5s 40 ¢ 3030 = C.3 20
In RESULTS AND DISCUSSION 40 50 3 30 10 20 The data in Fig. 1 are representative of a series Time, Hours of studies designed to show the level of phos1. Growth of A. vinelandii in phosphate-sufFIG. phate required to support a limited amount of ficient media (461 mg of P04 per liter) (0) and in growth of A. vinelandii under the conditions of phosphate-deficient media (4.61 mg of P04 per liter) these experiments. This limiting level of phos- (0). Solid lines indicate viable cell number and brophate (1/100 the amount in Burk medium) was ken lines indicate cell dry weight.
PHOSPHATE-LIMITED CULTURE OF A. VINELANDII
VOL. 139, 1979
641
TABLE 1. Effect ofphosphate deficiency on some cell constituents of A. vinelandii at late log phase (24 h) % cell dry Wta
Phosphate-deficient medium
Phosphate-sufficient control
Culture time (h)
PHB Nucleic acid 15 8.0 54 0 14 10.0 50 53 6 12 11.0 53 6 13.0 52 12 24 13.5 47 10 12.5 48 18 26 13.0 51 14 12.3 53 24 28 12.8 51 8 11.5 54 30 28 12.0 49 8 10.0 49 36 30 11.0 49 10 10.5 52 42 a 10 ml of with started were cultures the test inoculum culture; These measurements were obtained from the Protein
Nucleic acid
PHB
51b
9.0 12.0
14 6
Protein
inoculum. b All figures are the average of three separate determinations. TABLE 2. Effect ofphosphate on the amount of DNA and RNA in cells of A. vinelandii at late log phase (24 h) meAmt of phosphate in me
p(mg/litter)
dium dium (m/liter) 461 (control) 230 46 23 4.6
400-
Amt of cell Amt of cell DNA (% cell RNA (% cell dry wt) dry wt) 8.2 4.8 10.9 4.8 9.4 4.9 9.3 4.8 9.0 3.8
.
-
I-
-0. _
TI I
co
200
C-
Ea \a
Nt.,% I..
114,
1.4--%--44=4:-
-
0
50 40 30 20 Time, Hours FIG. 2. Phosphate content of the growth media (solid lines; in milligrams of P04 per liter) and cells (broken lines; in micrograms of P04 per milligram of protein) of A. vinelandii grown in phosphate-sufficient media (0) and phosphate-deficient media (0). U0
10
cultures were examined microscopically, pleomorphism was more marked than in control cultures. There was also more cell debris and many more spheroplasts in phosphate-limited cultures than in control cultures during late stationary phase (42 h). When these were examined by electron microscopy, it became evident that there were many cells with abnormal cell walls (Fig. 3).
Also, cells grown under phosphate-limited conditions contained greater amounts of PHB than did the control cells (Table 1). It is well established that glucose metabolism normally proceeds by amphibolic pathways in which the carbohydrate substrate is either shunted to biosynthesis reactions or is oxidized via the cytochrome system to regenerate ATP (14, 15). Organisms capable of storing excess substrate (5, 6) possess an additional choice in metabolic routing. When A. vinelandii is grown in phosphatedeficient media, excess unoxidized substrate is routed to PHB (25) via a pathway which does not involve phosphate directly. Since phosphate is essential for conversion of substrate energy to the ATP form, it seems logical that the pathway leading to ATP regeneration would be closed or restricted due to lack of inorganic phosphate. In this organism, the bifurcation in pathways is at the level of phosphoenolpyruvate (14, 15); if both the substrate pathway leading to biosynthetic reactions and that leading to ATP regeneration are inhibited by lack of inorganic phosphate (22, 27), then the entire substrate load would be channeled to PHB synthesis (Fig. 4). Since cells grown in phosphate-deficient media could have stopped growing for many reasons other than lack of phosphate, and since conversion from the vegetative cell to the cyst form probably required energy in the form of ATP (i.e., a high adenylate energy charge), biosynthesis could be assessed by inducing encystment. Table 3 shows that encystment in phosphatedeficient media was greatly reduced. In addition, regeneration of ATP in cells grown in phosphate-deficient media was considerably lower than that in cells grown in phosphate-sufficient control cultures (Table 4). Also, phosphate-deficient cells contained much larger amounts of stored PHB than did control cells (Table 1).
642
TSAI, ALADEGBAMI, AND VELA
J. BACTERIOL.
FIG. 3. Effect of phosphate deficiency on the morphology of A. vinelandii. (A) Normal cells. (B) Cells grown in phosphate-deficient media. Arrows indicate differences in cell wall appearance. x20,000.
These three observations are in agreement with the scheme suggested in Fig. 4. Previous investigators (3, 4, 13, 33) have claimed that oxygen uptake in phosphate-limited cultures is inhibited because all available
nucleotide phosphate goes to ATP. This is probably not so. It is difficult to accept the idea proposed by Dalton and Postgate (3, 4) because this is a reaction which requires the presence of readily available inorganic phosphate in the me-
PHOSPHATE-LIMITED CULTURE OF A. VINELANDII
VOL. 139, 1979
Glucose
Phosphoenol-pyruvate
t
Pyruvate III
PHB
High
Adenylate Charge
X
,
Ac-S-CoA Low
4
,,Adenylate Charge
" -Ketoglutarate/Malate
a""11 BIOSYNTHESIS '
I ADP-ATP
Phosphate Deficiency FIG. 4. Pathways for substrate energy utilization in A. vinelandii. Roman numerals indicate priority and x's indicate the inhibiting or "switching" condition. Ac-S-CoA, Acetyl coenzyme A. TABLE 3. Effect ofphosphate deficiency on cyst formation in A. vinelandii Encystment (days)
1 2 3 4 5 6 7
Phosphate-sufficient control Total no. of viable cells % Cysts and cysts
1.4 x 108 2.5 x 108 3.8 x 108 5.7 x 108 5.6 x 108 3.4x108 5.2 x 108
5 15 45 68 86 96 98
Phosphate-deficient culture Total no. of viable cells Cysts and cysts 1.1 X 108 0
6.2 x 1.1 x 1.2 x 1.3 X
108
108 105 108 3.2x108 2.3 x 108
0 3 7 8 9 9
643
dium. The contention that cell death in phosphate-deficient cultures is due to a high ratio of ATP to ADP (4) or to low ratios of ADP to ATP (13) was not supported by laboratory studies. We performed measurements of the adenylate nucleotides and showed that the reverse of the explanation offered by Postgate and his co-workers (3, 4, 13) is actually true. Table 4 shows that the energy charge of cells grown in phosphatedeficient media was slightly lower than that of the control cells. This alone would be sufficient reason for doubting that cells in phosphate-limited cultures die because the ratio of ATP to ADP becomes too high and inhibits respiration. Although this may be a mechanism well recognized in eucaryotic cells (8, 12), our data show that it is not one operative in Azotobacter. Although it has been reported that respiration in A. chroococcum is controlled by adenine nucleotides, no actual measurements of adenine nucleotides were given to support the conclusions presented in that study (33). In this report, direct verification is given in the form of ATP-ADP ratios. First, the ratio of ATP to ADP is much greater in the control cells than in cells grown in
phosphate-deficient media. In the earlier reports (3, 4) it was postulated that oxygen uptake was inhibited because some high value of the ratio of ATP to ADP was attained. Quite to the contrary, cultures actively consuming oxygen have much higher ratios of ATP to ADP than do those which have stopped (Table 5). The data presented here suggest that the high ratios of ATP to ADP play no directly discernible role in the rate of oxygen uptake in cultures of A. vinelandii. Clearly then, it is not the ratio of ATP to ADP which inhibits respiration in the cells of A. vinelandii. Under conditions of phosphate limitation and sufficient oxygen and glucose (Table 5), AMP is converted to ADP; this
TABLE 4. Distribution of adenine nucleotides, adenylate charge, and the ratio of ATP to ADP in vegetative cells of A. vinelandii grown in phosphate-deficient media Phosphate-deficient medium
Phosphate-sufficient control Time (h)
0 6 12 18 24 30 36 42 48
ATP concn"
ADP concn"
AMP concn"
Energy
Ratio of
charge'
ADP
3.44 11.01 16.54 13.50 15.23 8.86 11.74 10.20 9.58
2.60
JOURNAL OF BACTERIOLOGY, Aug. 1979, p. 639-645 0021-9193/79/08-0639/07$02.00/0
Phosphate-Limited Culture of Azotobacter vinelandii J. C. TSAI, S. L. ALADEGBAMI, AND G. R. VELA* Department of Biological Sciences, North Texas State University, Denton, Texas 76203
Received for publication 16 May 1979
Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly,8-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.
Although the Azotobacteriaceae have been extensively studied for almost 100 years (7, 11, 24, 32) and although it has long been known that phosphorous (phosphate) is a vital element in all living cells, little is known of the effect of phosphate deprivation on the physiology of these bacteria. Although the role of phosphate in the structure and function of other microorganisms has been studied in great detail (9, 20, 23) only a few reports regarding specific effects of phosphate limitation on Azotobacter appear in the literature (3, 4, 13, 19). Of these, the work which describes the loss of cell viability in phosphate-deficient cultures of Azotobacter chroococcum (3, 4, 13) raises provocative and important questions. Dalton and Postgate (3, 4) reported growth inhibition and decreased viability in phosphate. limited cultures and found a correlation between cellular oxygen consumption and nitrogenase function. This led them to the conclusion that phosphate-limited cultures stop growing in the presence of oxygen because ADP is converted to ATP and the resulting increased ratio of ATP to ADP "shuts off" respiration. They further postulated that this allows oxygen to accumulate in the cell, causing the inactivation of the nitrogenase system. Lees and Postgate (13) reiterated this explanation and offered supporting evidence' by showing that phosphate-sufficient cells remove dissolved oxygen from culture fluid, whereas phosphate-deficient cells fail to do so under the same conditions. From these reports, it appears that decreased viability of cells grown in phosphate-deficient media was due to oxidation of nitrogenase, which was a consequence of the phosphorylation of ADP.
This is a report on measurements of the adenylate pools in cells of Azotobacter vinelandii grown in phosphate-deficient media. The experiments were conducted in batch cultures rather than chemostat cultures since the former show better the effect of phosphate limitation during different physiological states of the organism in question. Also, the conditions of the experiments, i.e., vigorous aeration with consequent maximal oxygenation of the culture medium, permitted us to distinguish the effects of phosphate deficiency from those of oxygen toxicity. A recent report (10) seriously questions the idea of oxygen toxicity and, in this respect, is in agreement with some of the data reported here. MATERIALS AND METHODS
Cultures. Stock cultures of A. vinelandii ATCC 12837 were maintained on Burk nitrogen-free agar slants (31) and were streaked onto plates or inoculated into liquid media when needed. Burk medium contained 640 mg of K2HPO4 and 160 mg of KH2PO4 per liter of water. Phosphate-deficient media were prepared with only 6.4 mg of K2HPO4 and 1.6 mg of KH2PO4 per liter of water. Normal or phosphate-deficient encystment media were prepared by replacing glucose with filter-sterilized n-butanol to a final concentration of 0.3%. Comparisons between normal cells and those grown in phosphate-deficient media were made from cultures started with identical inocula from the same seed culture. The pH's of both normal and low-phosphate cultures were periodically monitored and adjusted to 7.3 by adding 0.1 N KOH. All cultures were grown at 30°C, and liquid cultures were incubated with shaking to insure adequate aeration, i.e., a residual oxygen level of more than 1 mg of dissolved 02 per liter. Chemical determinations. The protein content of both control and phosphate-deficient cells was 639
640
TSAI, ALADEGBAMI, AND VELA
measured by the method of Murphy and Kiew (18) and also by the method of Warburg and Christian (30). The two were run simultaneously for comparison. The Warburg-Christian method (30) for nucleic acid assay was used throughout these studies, and the procedure described by Senior et al. (25) was used for all determinations of poly-f,-hydroxybutyric acid (PHB). The method of Bligh and Dyer (1) was used for determination of total cell lipids, and phosphates were measured by the stannous chloride method described by Sletten and Bach (26). Adenylate charge. The procedure suggested by Chapman et al. (2) was used for measuring ATP, ADP, and AMP and also for calculating the energy charge of the adenylate pool. There were, however, two significant departures from the published method: (i) 50 mg of buffered firefly lantern extract (Sigma Chemical Co., St. Louis, Mo.) was reconstituted in 5.0 ml of deionized water, stored at -20°C overnight, and filtered through a glass fiber pad before use; and (ii) a model 2000 integrating photometer (SAI Scientific Co., San Diego, Calif.) was used in all measurements. Reference curves for ATP were linear and reproducible in the range of 10-12 to 10-`5 mol of ATP in the reaction mixture. All other enzymes and substrates required for these assays were obtained from Sigma Chemical Co. Respiratory activities. Oxygen uptake of whole cells was determined at 30°C in a Gilson model KlC Oxygraph with a Clark-type electrode (Gilson Medical Electronics, Inc., Middleton, Wis.).
Encystment studies. Cultures of A. vinelandii were grown to late log phase in phosphate-sufficient and phosphate-deficient media. The cells were washed twice by centrifugation and plated on encystment media containing the same amount of phosphate as the growth medium. Cultures were examined for cyst formation by a previously described staining procedure
J. BACTERIOL.
chosen from preliminary studies because it yielded approximately 60% the number of cells found in control cultures. The growth rate during the early log phase was essentially the same as in control cultures, although the lag phase was slightly longer. Further studies showed that cells grown in phosphate-deficient media contained approximately the same amount of nucleic acid and protein as did cells grown in phosphate-sufficient media (Table 1). Another study (Table 2) showed that the amount of phosphate in the medium had only a minor effect on cell DNA and RNA. These data imply that cells grown in phosphate-deficient media were different from control cells in some respects but not in others. For example, although the medium designated phosphate-deficient contained only 1/100 the amount of phosphate present in the control medium, the amount of phosphate in the cells from the two cultures was essentially the same (Fig. 2). It can be inferred from the data in Fig. 2 that cell division was accomplished by phosphate sharing and that each daughter cell received an adequate allotment of available phosphate. When the total amount fell below a given level, cell division ceased. It appears certain, then, that these were normal cells adapted to a growth environment where the dearth of phosphate affected (determined) the total cell yield of the cultures but had little effect on other aspects of cell growth, such as total protein and nucleic acids. On the other hand, when phosphate-limited
(29).
Electron microscopy. Cells were harvested by centrifugation, washed three times in distilled water, and suspended in 3% glutaraldehyde buffered with 0.1 M sodium cacodylate. They were fixed for 1 h as described by Vela et al. (28), washed again, and then placed in 1% (vol/vol) OS04 in cacodylate buffer at 4°C for 1 h. They were again washed and then dehydrated by passing through 30, 50, 75, 85, 95, and 100% (vol/vol) ethanol solutions before embedding in Epon 812. Thin sections were cut with a Porter-Blum ultramicrotome (Ivan Sorvall, Inc., Norwalk, Conn.) equipped with a glass knife. All sections were stained with uranyl acetate for 45 min and then with tartrate for 30 min and examined with an RCA-EMU-3G electron microscope at initial magnifications of x11,500 to x16,000.
9.5 r
9.0 .5a 'a z
8.5 h 0'
/
8.0 -,1 '
80 70 -E 60 U 50 :L .5s 40 ¢ 3030 = C.3 20
In RESULTS AND DISCUSSION 40 50 3 30 10 20 The data in Fig. 1 are representative of a series Time, Hours of studies designed to show the level of phos1. Growth of A. vinelandii in phosphate-sufFIG. phate required to support a limited amount of ficient media (461 mg of P04 per liter) (0) and in growth of A. vinelandii under the conditions of phosphate-deficient media (4.61 mg of P04 per liter) these experiments. This limiting level of phos- (0). Solid lines indicate viable cell number and brophate (1/100 the amount in Burk medium) was ken lines indicate cell dry weight.
PHOSPHATE-LIMITED CULTURE OF A. VINELANDII
VOL. 139, 1979
641
TABLE 1. Effect ofphosphate deficiency on some cell constituents of A. vinelandii at late log phase (24 h) % cell dry Wta
Phosphate-deficient medium
Phosphate-sufficient control
Culture time (h)
PHB Nucleic acid 15 8.0 54 0 14 10.0 50 53 6 12 11.0 53 6 13.0 52 12 24 13.5 47 10 12.5 48 18 26 13.0 51 14 12.3 53 24 28 12.8 51 8 11.5 54 30 28 12.0 49 8 10.0 49 36 30 11.0 49 10 10.5 52 42 a 10 ml of with started were cultures the test inoculum culture; These measurements were obtained from the Protein
Nucleic acid
PHB
51b
9.0 12.0
14 6
Protein
inoculum. b All figures are the average of three separate determinations. TABLE 2. Effect ofphosphate on the amount of DNA and RNA in cells of A. vinelandii at late log phase (24 h) meAmt of phosphate in me
p(mg/litter)
dium dium (m/liter) 461 (control) 230 46 23 4.6
400-
Amt of cell Amt of cell DNA (% cell RNA (% cell dry wt) dry wt) 8.2 4.8 10.9 4.8 9.4 4.9 9.3 4.8 9.0 3.8
.
-
I-
-0. _
TI I
co
200
C-
Ea \a
Nt.,% I..
114,
1.4--%--44=4:-
-
0
50 40 30 20 Time, Hours FIG. 2. Phosphate content of the growth media (solid lines; in milligrams of P04 per liter) and cells (broken lines; in micrograms of P04 per milligram of protein) of A. vinelandii grown in phosphate-sufficient media (0) and phosphate-deficient media (0). U0
10
cultures were examined microscopically, pleomorphism was more marked than in control cultures. There was also more cell debris and many more spheroplasts in phosphate-limited cultures than in control cultures during late stationary phase (42 h). When these were examined by electron microscopy, it became evident that there were many cells with abnormal cell walls (Fig. 3).
Also, cells grown under phosphate-limited conditions contained greater amounts of PHB than did the control cells (Table 1). It is well established that glucose metabolism normally proceeds by amphibolic pathways in which the carbohydrate substrate is either shunted to biosynthesis reactions or is oxidized via the cytochrome system to regenerate ATP (14, 15). Organisms capable of storing excess substrate (5, 6) possess an additional choice in metabolic routing. When A. vinelandii is grown in phosphatedeficient media, excess unoxidized substrate is routed to PHB (25) via a pathway which does not involve phosphate directly. Since phosphate is essential for conversion of substrate energy to the ATP form, it seems logical that the pathway leading to ATP regeneration would be closed or restricted due to lack of inorganic phosphate. In this organism, the bifurcation in pathways is at the level of phosphoenolpyruvate (14, 15); if both the substrate pathway leading to biosynthetic reactions and that leading to ATP regeneration are inhibited by lack of inorganic phosphate (22, 27), then the entire substrate load would be channeled to PHB synthesis (Fig. 4). Since cells grown in phosphate-deficient media could have stopped growing for many reasons other than lack of phosphate, and since conversion from the vegetative cell to the cyst form probably required energy in the form of ATP (i.e., a high adenylate energy charge), biosynthesis could be assessed by inducing encystment. Table 3 shows that encystment in phosphatedeficient media was greatly reduced. In addition, regeneration of ATP in cells grown in phosphate-deficient media was considerably lower than that in cells grown in phosphate-sufficient control cultures (Table 4). Also, phosphate-deficient cells contained much larger amounts of stored PHB than did control cells (Table 1).
642
TSAI, ALADEGBAMI, AND VELA
J. BACTERIOL.
FIG. 3. Effect of phosphate deficiency on the morphology of A. vinelandii. (A) Normal cells. (B) Cells grown in phosphate-deficient media. Arrows indicate differences in cell wall appearance. x20,000.
These three observations are in agreement with the scheme suggested in Fig. 4. Previous investigators (3, 4, 13, 33) have claimed that oxygen uptake in phosphate-limited cultures is inhibited because all available
nucleotide phosphate goes to ATP. This is probably not so. It is difficult to accept the idea proposed by Dalton and Postgate (3, 4) because this is a reaction which requires the presence of readily available inorganic phosphate in the me-
PHOSPHATE-LIMITED CULTURE OF A. VINELANDII
VOL. 139, 1979
Glucose
Phosphoenol-pyruvate
t
Pyruvate III
PHB
High
Adenylate Charge
X
,
Ac-S-CoA Low
4
,,Adenylate Charge
" -Ketoglutarate/Malate
a""11 BIOSYNTHESIS '
I ADP-ATP
Phosphate Deficiency FIG. 4. Pathways for substrate energy utilization in A. vinelandii. Roman numerals indicate priority and x's indicate the inhibiting or "switching" condition. Ac-S-CoA, Acetyl coenzyme A. TABLE 3. Effect ofphosphate deficiency on cyst formation in A. vinelandii Encystment (days)
1 2 3 4 5 6 7
Phosphate-sufficient control Total no. of viable cells % Cysts and cysts
1.4 x 108 2.5 x 108 3.8 x 108 5.7 x 108 5.6 x 108 3.4x108 5.2 x 108
5 15 45 68 86 96 98
Phosphate-deficient culture Total no. of viable cells Cysts and cysts 1.1 X 108 0
6.2 x 1.1 x 1.2 x 1.3 X
108
108 105 108 3.2x108 2.3 x 108
0 3 7 8 9 9
643
dium. The contention that cell death in phosphate-deficient cultures is due to a high ratio of ATP to ADP (4) or to low ratios of ADP to ATP (13) was not supported by laboratory studies. We performed measurements of the adenylate nucleotides and showed that the reverse of the explanation offered by Postgate and his co-workers (3, 4, 13) is actually true. Table 4 shows that the energy charge of cells grown in phosphatedeficient media was slightly lower than that of the control cells. This alone would be sufficient reason for doubting that cells in phosphate-limited cultures die because the ratio of ATP to ADP becomes too high and inhibits respiration. Although this may be a mechanism well recognized in eucaryotic cells (8, 12), our data show that it is not one operative in Azotobacter. Although it has been reported that respiration in A. chroococcum is controlled by adenine nucleotides, no actual measurements of adenine nucleotides were given to support the conclusions presented in that study (33). In this report, direct verification is given in the form of ATP-ADP ratios. First, the ratio of ATP to ADP is much greater in the control cells than in cells grown in
phosphate-deficient media. In the earlier reports (3, 4) it was postulated that oxygen uptake was inhibited because some high value of the ratio of ATP to ADP was attained. Quite to the contrary, cultures actively consuming oxygen have much higher ratios of ATP to ADP than do those which have stopped (Table 5). The data presented here suggest that the high ratios of ATP to ADP play no directly discernible role in the rate of oxygen uptake in cultures of A. vinelandii. Clearly then, it is not the ratio of ATP to ADP which inhibits respiration in the cells of A. vinelandii. Under conditions of phosphate limitation and sufficient oxygen and glucose (Table 5), AMP is converted to ADP; this
TABLE 4. Distribution of adenine nucleotides, adenylate charge, and the ratio of ATP to ADP in vegetative cells of A. vinelandii grown in phosphate-deficient media Phosphate-deficient medium
Phosphate-sufficient control Time (h)
0 6 12 18 24 30 36 42 48
ATP concn"
ADP concn"
AMP concn"
Energy
Ratio of
charge'
ADP
3.44 11.01 16.54 13.50 15.23 8.86 11.74 10.20 9.58
2.60
