JOURNAL OF BACTERIOLOGY, Nov. 1977. p. 520-525 Copyright 1977 American Society for Microbiology
Vol. 132, No. 2 Printed in U.S.A.
Adaptive Changes in Phosphate Uptake by the Fungus Neurospora crassa in Response to Phosphate Supply ROSS E. BEEVER* AND DONALD J. W. BURNS Plant Diseases Diision, Departmett of Scientific and Industrial Research, Auckland, Newl Zealand Received for publication 17 August 1977
The phosphate uptake rate of Neurospora crassa germlings growing exponentially in media containing phosphate at concentrations between 10 mM and 50 ,uM was virtually constant. The uptake characteristics of these germlings were studied in detail assuming the simultaneous operation of two uptake systems, one of low affinity and one of high affinity. The K,,, of the low-affinity system was constant after growth at phosphate concentrations greater than 1 mM but became progressively lower as the concentration was reduced below 1 mM. In contrast, the K,,, of the high-affinity system was independent of the phosphate concentration of the growth medium. The V,, ,,' of each system was highest after growth at low phosphate concentrations. As the phosphate concentration was increased to a maximum of 100 mM, the V,,,,,,. of the low-afflnity system fell gradually, whereas that of the high-affinity system at first fell rapidly but then reached a constant minimum value at concentrations of 2.5 mM and higher. The differences in the kinetic parameters fully account for the constancy of uptake rate shown by the germlings. There have been many studies of the kinetics of solute uptake systems, and in many instances uptake characteristics have been shown to differ in similar cells which have been exposed to different environments before study. However, there seems to be no instance where the activity of an uptake system(s) in an organism growing under a range of conditions has been precisely related to the requirements of the organism for the solute under those conditions. Such quantitative studies are needed to further our understanding of how uptake systems are controlled. As part of such a study, we have shown that phosphate uptake by Neurospora crassa germlings after growth at either a high (10 mM) or low (50 ,uM) phosphate concentration is due to the simultaneous operation of two uptake systems (1). In the present paper we show that, at the time of study, the growth rate and the rate of phosphate entry of such germlings are independent of the phosphate concentration in the growth medium. The observation made previously (1), that the kinetic parameters of the uptake systems are influenced by the growth phosphate concentration, is extended to germlings growing at various concentrations within the range 50 ,.M to 100 mM. We find that the differences in the kinetic parameters are sufflcient to account precisely for the constancy of phosphate entry during growth. The results permit a detailed description of the way in which N. crassa adapts to phosphate supply.
MATERIALS AND METHOI)S The strain of fungus used and the growth conditions, including the composition of the phosphatefree base medium, have been described (1). For convenience, germlings are referred to by the initial phosphate concentration of the medium in which they were grown (e.g., 50 ,uM germlings were germinated and grown in medium initially containing 50 ,uM phosphate). The slight decreases in concentration that occur during growth have been taken into account where appropriate in calculations and graphs. The time of inoculation is taken as zero time. Estimates of doubling times (t,,) were calculated using least-squares analysis. Where appropriate, measurements are given as the mean + 1 standard deviation. I)ry weight and total cell phosphorus determinations. To determine dry weights, germlings (usually from 500 ml of culture) were harvested onto preweighed cellulose nitrate filters (1.2-,.cm pore size) and washed twice with water. The filter and pad of germlings were dried to constant weight under vacuum over P205. For total cell phosphorus determination, the dried pad and filter were acid-digested. and the phosphate content of the digest was measured (3). Uptake rate measurements with 32p,. Unless otherwise specified, uptake rate measurements were made with our standard method (1), which involves harvesting, washing, and resuspending germlings in fresh medium containing 14 ,M cycloheximide and with 32P, at the chosen concentration. In one experiment (Fig. 4), we also measured uptake with a modified method, which avoids harvesting or washing the germlings and in which cycloheximide is not present in the uptake solution. The procedure
520
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was as follows: at 2 h 20 min, 19 ml of medium containing germlings was transferred from the large growth flask to a smaller flask; at 2 h 26 min, 1 ml of 32Pi-labeled medium of the same phosphate concentration as the growth medium was added. The phosphate uptake rate over the next 8 min was determined from 1-ml samples removed at 1-min intervals. Shaking was continued, and the temperature was maintained at 30°C throughout. Calculation of uptake and estimation of kinetic parameters. It was shown previously (1) that uptake in N. crassa can be accounted for by the operation of two systems, each obeying Michaelis-Menten kinetics. This relationship is expressed by the equation for the double hyperbola:
Vmar HA) S Km(LA) S K",(HA, + S where v is uptake rate; s is phosphate concentration; Vma,LA) and VmaJHA) are the maximum uptake rates of the low- and high-affinity systems, respectively; and K,f(LA) and Km(HA) are the phosphate concentrations that give rise to half-maximum uptake rates for each system. This equation has been used as follows. (i) Calculation of uptake rates: where the values of all four kinetic parameters were known for a given type of germling, the uptake rate at any chosen phosphate concentration was calculated directly. (ii) Estimation of Km values: for a given germling preparation, uptake rates were measured at 15 phosphate concentrations, over a wide concentration range, and a double-hyperbola equation was fitted to the data as described previously (1). (iii) Estimation of V values: the method used to obtain Km values also gives Vnax values, but, because of the large number of uptake measurements required, it was not possible in any one such experiment to measure V nax values for more than two germling types. Vmax values reported in this paper (Fig. 3 and 6) were therefore obtained by using a shorter method that depends on knowing the values for Km(LA) and Km(HA) (Fig. 5). Briefly, for each germling type, uptake rates measured at 1 mM and 10 ,uM phosphate were substituted in turn into the double-hyperbola equation. The resulting two simultaneous equations were then solved for Vmaj.LA) and Vmax,HA) after inserting the Km values. VfnalG8IA) v=--
+
S
+
103
E
102
10
2
0
6 TIME ( h
4
8
10
FIG. 1. Effect of phosphate concentration in the medium on growth of N. crassa. Conidia were inoculated at zero time into flasks containing base medium plus 10 mM (C]), 50 ELM (0), or zero (A) phosphate. Each point represents the harvest from one flask. 80 70 60 0
2
50
X~~
D
40
a 2.
110 0 E E z 30
,;:X
0. -
o m uLU
3:-4
>- I.cc0
13
20
F
J.-
8
IV
3.5
2.5
4.5
TIME ( h)
FIG. 2. Changes in dry weight and total cell phosphorus during growth over 2.5 to 4.5 h. Conidia were inoculated at zero time into flasks of base medium containing 10 mM or 50 /LM phosphate. Dry weight (10 mM germlings [I], td = 2.09 h; 50 .M germlings [0], td = 2.19 h) and total cell P (10 mM germlings [E], td 1.89 h; 50 MM germlings [0], td = 1.81 h) were determined at intervals. Each point represents the mean value of single harvests from two independent experiments. =
RESULTS Growth studies. In a preliminary experiment it was found that increase in dry weight of germlings at a high phosphate concentration (10 mM) became exponential by 2.5 h and time to compare the uptake behavior of germcontinued in exponential fashion at least until lings growing at different phosphate concentra10 h (Fig. 1). If no phosphate was present in tions. At this time exponential growth, as the growth medium, the germling dry weight judged by dry-weight increase, had comfollowed that obtained with the high-phosphate menced, but the phosphate concentration of treatment until about 3 h. If a low concentra- the medium had altered little; for medium tion of phosphate (50 AM) was present, the containing 10 mM phosphate the fall was neggermling dry weight followed that obtained ligible, and for the medium containing 50 ,uM with the high-phosphate treatment until about phosphate the concentration had dropped to 6 h. We chose 2.5 h as the most appropriate about 44 ,M. More detailed study (Fig. 2;
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unpublished data) confirmed that, over the period 2.5 to 4.5 h, the dry-weight increases of 10 mM and 50 ,uM germlings were the same. By 2.5 h, 50 to 75% of the conidia had produced germ tubes; by 3.5 h over 90% had produced germ tubes. Although the dry weights of the cultures showed an exponential increase from 2.5 h, it does not necessarily follow that all growth parameters were increasing exponentially by this time. We found that although total cell phosphorus at 2.5 h differed slightly between the two types of germlings (Table 1), in each case the rate of its increase per unit volume of culture
01 E
EL
01
was
volume of culture basis so that a direct assessment of the growth-dependent increases in uptake activity for each system could be made. (Elsewhere we have followed the normal practice of expressing V,,,,avalues in terms of tissue dry weight.) Phosphate uptake rates at the concentration used for growth. From knowledge of the total cell phosphorus content at 2.5 h, and the doubling time of this parameter, one can calculate the phosphorus uptake rate of the germlings at 2.5 h. Table 1 shows that estimates of the phosphorus uptake rate for 10 mM and 50 FLM germlings calculated from such data were very similar despite the 200-fold difference in growth phosphate concentration. Because phosphate was the only phosphorus source supplied in the medium and there was no significant phosphorus efflux during uptake (1), we can equate phosphorus uptake measured in this way with phosphate uptake. A less laborious method of determining phosphate uptake at the growth concentration is to measure uptake using 32p;. This was done, by our standard method, for germlings growing at seven phosTABLE 1. Uptake rate estimates based on total cell phosphorus measurements Phosphate
in
Total cell P
germation
at 2.5 h'
medium
cmwIt])
10 mM 50 ,M
558 + 30 507 29
germinationy [dry
Duln paert Doubling Uptake ratet for cell (~tm ol/g [dlry pb (h) wt] per min)
001 25
35 TIME h
45
FIG. 3. Changes in the V ,,,a values (expressed on per-unit-volume of culture basis) of the two phosphate uptake systems during growth ouer 2.5 to 4.5 h. V ,,ax values were calculated from uptake rate a
measurements made at 1 mM and 10 pM phosphate (see text). Symbols: (A) 10 mM germlings-O-, lowaffinity system; *, high-affinity system. (B) 50 ,uM germlings- 0, low-affinity system; 0, high-affinity
system.
E -1
4
-0 E _ 2
CL
10-
PHOSPHATE IN GROWTH MEDIUM
M )
FIG. 4. Effect of phosphate concentration of the growth medium on the phosphate uptake rate. N. crassa germlings were grown at phosphate concentrations from 50 ,M to 10 mM. At 2.5 h, uptake rates at the respective growth phosphate concentrations were measured by either our standard method (a) or the modified method (0). The dotted line is the mean uptake rate (standard method) for all treatments.
tim e
1.89 1.81
3.41 3.24
0.18 0.19
Mean of means of three experiments. on the experiments of Fig. 2. Calculated from the equation: uptake rate = (cell P x ln 2)/doubling time. The standard deviations given are minimum estimates based on the total cell phosphorus measurements only. "
1.0
E
exponential (Fig. 2). The V,/iax values of the uptake systems of both types of germlings were also found to increase in exponential fashion (Fig. 3). In this instance the Vii,ax values have been expressed on a per-unit-
bBased
I
0.01
_
phate concentrations between 10 mM and 50 ,tM (Fig. 4). Uptake rates by this standard procedure were very similar (mean, 3.21 + 0.20 tmol/g [dry weight] per min) irrespective of the phosphate concentration of the growth medium. Use of the modified uptake assay method, which disturbs the germlings less, gave identical results (mean, 3.23 + 0.22 ,umol/ g [dry weight] per min; Fig. 4).
PHOSPHATE UPTAKE IN NEUROSPORA
VOL. 132, 1977
value for 1 mM germlings was close to that obtained for 10 mM germlings, whereas that for 200 ,uM germlings was about midway between the values for 50 ,uM and 1 mM germlings. The K,,,(HA) values of both 200 ,uM and 1 mM germlings were within the range of the values obtained for 50 ,uM and 10 mM germlings. Since the Km HA) values of 10 mM and 50 ,uM germlings were not significantly different (1), we conclude that K,l,HAI is constant (2.75 + 0.34 ,uM, eight determinations) in germlings grown in phosphate concentrations between 50 uM and 10 mM. The Vmar values of 2.5-h germlings grown at seven phosphate concentrations are shown in Fig. 6A. Also shown are the calculated uptake rates, based on these V,,,ar values, for germlings at their respective growth concentrations. Although the calculated rates of uptake were slightly higher at the higher growth concentrations, the mean value of 3.14 + 0.25 ,umol/g (dry weight) per min was close to the mean uptake rate obtained by direct measurement. In an attempt to find whether the high-affinity system could be eliminated completely from germlings, we grew them at very high phosphate concentrations (25 and 100 mM). The uptake rates of these germlings at 10 ,uM and 1 mM phosphate concentrations were close to those of 10 mM germlings. If the assumption is made that Km(LA, and K,,,HA, are the same in 25 mM and 100 mM germlings as in 10 mM germlings, then the Vinax values can be estimated. Values derived in this manner indicate
Differences in the uptake systems after growth at different phosphate concentrations. In the previous paper (1) it was shown that some kinetic parameters of the uptake systems of 10 mM and 50 ,uM germlings were different. In the present paper we have determined K,M, values for germlings growing at 1 mM and 200 pAM phosphate (Fig. 5). The Km(LA) A
1200
0
900
600
-300
u
E I
111
1
,,.
1
B
2~
i0-4
10-3
10-2
PHOSPHATE IN GROWTH MEDIUM ( M )
FIG. 5. Effect of phosphate concentration of the growth medium on Km values of the uptake systems. (A) Low-affinity system; (B) high-affinity system. Each point represents an individual determination of K,1. The solid line through the Km values of the low-affinity system was fitted by inspection. The dotted line through the K,m values of the high-affinity system is the mean of all determinations irrespective of growth concentration. Data for germlings grown at 10 mM and 50 ,uM are from the accompanying paper (1). E
523
B
A
6
Z4
io4
10 -3
o -3 10 -2 PHOSPHATE IN GROWTH MEDIUM (M)
10-2
io -
FIG. 6. Effect of phosphate concentration of the growth medium on V,ax values of the uptake systems. Results from two experiments are shown. (A) Growth phosphate concentrations of 10 mM and below; (B) growth phosphate concentrations of 2.5 mM and above. V,max values were calculated from uptake rate measurements made at 10 IAM and 1 mM phosphate (see text). In (A) Km(HA) was taken as 2.75 tAM and K,m(LA) values were read from Fig. 5. In (B) Km(LA) and Km(HA) were assumed to be the same as for 10 mM germlings. In addition, these kinetic values were used to calculate the uptake rates of the germlings at their respective growth concentrations. The solid lines through the Vmax values have been fitted by inspection; the dotted lines through the calculated uptake values are the means of all values in each experiment. Symbols: 0, Vma,lHA); A, calculated uptake at the growth concentration. V111aZLA);
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BEEVER AND BURNS loo
these small differences in doubling time, it is apparent that growth of the cultures is approximately balanced under the conditions of study. 60 , F Thus, we suggest that results obtained with such germlings can be taken as representative of mycelium at later stages of exponential growth. Changes in the parameters of the uptake O §sWslw Ts ,§TTT ...IT IK systems. The constancy of phosphate uptake 10' rate over a widt concentration range (Table 1, FIG. 7. Diagram showing the relative contribu- Fig. 4) shows that N. crassa exerts a tight control over the activity of its uptake systems. tions of the low- and high-affinity systems to phosphate uptake, at the growth concentration, of 2.5-h Previously, we have established that the kigermlings growing at different phosphate concentra- netic parameters of the uptake systems can tions. Calculated from the data of Fig. 5 and 6A differ depending on the phosphate concentraand B. tion of the growth medium (1). The calculated uptake rates presented in Fig. 6, which are that the high-affinity system does not change based on such kinetic analyses, show that changes in these parameters can account for as the growth phosphate concentration is increased above 10 mM, but the VM,Xr of the low- the observed constancy of uptake. Thus, the affinity system falls (Fig. 6B). The predicted mechanism by which N. crassa adapts to phosuptake rates during growth, calculated using phate supply can be described in detail. At the respective parameter values, were rela- high phosphate concentrations in the growth tively constant over this concentration range medium, uptake is due predominantly to the (mean value, 3.54 + 0.32 ,amol/g [dry weight] low-affinity system [Km(IA,, about 1 mM], as at these concentrations Vm,a,n 1A is so low that the per min). Relative contributions of systems to up- contribution of the high-affinity system is less take. The relative contribution of each uptake than 10% in the total (Fig. 7). Although V,, a. A) system to uptake during growth at a given rises progressively as the growth phosphate phosphate concentration is summarized in Fig. concentrations is lowered from 100 to 1 mM 7. From this diagram it can be predicted that (Fig. 6A and B), this rise is counter-balanced the two systems would contribute equally to by the decreasing phosphate concentration, so uptake in germlings growing at 145 ,tM phos- that the amount contributed by this system to uptake remains relatively constant. At concenphate. trations lower than 1 mM the contribution of DISCUSSION the low-affinity system would decrease rapidly Growth studies. The growth habit of fila- if V,,,aLI,A) and K .,LIA, remained at the values mentous fungi makes them unsuitable for con- found in 1 mM germlings. However, the effectinuous-culture growth techniques. Therefore, tive activity of this system is increased both by a continuing increase in Vm,a,L.A\ to a plateau we chose to use batch culture techniques and study uptake during the period of exponential value at 200 ,uM and below (Fig. 6A), and a growth. Although the dry-weight increase was decrease in K,,,l,1A) (Fig. 5A). Although these exponential by 2.5 h, the dry weight at that changes increase the ability of the low-affinity time was still less than twice that of the inocu- system to mediate phosphate uptake, this syslum (Fig. 1 and 2). We felt it desirable, there- tem alone cannot account for the observed fore, to establish whether the growth parame- uptake rates, and, particularly at the lower ters relating to phosphate uptake were also phosphate concentrations, the high-affinity exponential by this stage. Figures 2 and 3 show system becomes relatively more important. that both the total cell phosphorus and the From a constant low value at phosphate conV,,,,(1 values for both phosphate uptake systems centrations greater than 1 mM, Vi,ar MA) in(expressed on a per-unit of culture basis) in- creases steadily as the concentration in the creased in exponential fashion over the period growth medium decreases, until at 50 tM 2.5 to 4.5 h. The doubling times obtained for VMaxfHA) is over eight times its value at 10 mM these parameters were consistently less than (Fig. 6A). In contrast to the low-affinity systhe doubling time for dry weight. The resultant tem, Km,,(I1A, is unaffected by the external phosslight increase in total cell phosphorus per unit phate concentration (Fig. 5B). of dry weight may reflect developmental In a similar study, also of N. crassa, Lowenchanges during the 2.5- to 4.5-h period. Despite dorf et al. (5. 6) attempted to correlate the v
0
80 _
-
2(20
-
4
z
z
8
40
6
20
_ 8(iO
-
-r
10 4C 2 PHOSPHATE IN GROWTH MEDIUM
"I
M
VOL. 132, 1977
PHOSPHATE UPTAKE IN NEUROSPORA
525
phosphate uptake rate predicted from the activ- type are produced during growth at low phosities of the uptake systems at the phosphate phate concentrations. A similar explanation concentration of the growth medium (37 mM cannot account for the changes in the lowin their case) with the phosphate uptake rate affinity system because K, ,A) as well as as determined from total cell phosphorus and Viazr1LA) can vary. However, such changes can doubling-time values. Using two wild-type be accounted for by models in which the intrastrains, they found that the respective uptake cellular concentration of either the transported rates predicted from kinetic studies were 1.36 substrate or a related metabolite can directly and 1.63 times the rates determined from meas- modify the activity of the uptake system. There urements of total cell phosphorus. These results are a number of precedents for such control of are in contrast to the close agreement we have an uptake system in fungi (2, 7). found. However, the uptake assay used by ACKNOWLED)GNIENT these workers has not been shown to give uptake rates comparable to ones measured Philippa Clark gave excellent technical assistance. without disturbing the fungus. It is possible that their assay procedure. which differs from LITERATURE CITED ours in a number of ways (1), alters the uptake 1. D. J. W., and R. E. Beever. 1977. Kinetic Burns, systems in some respect. characterization of the two phosphate uptake systems There is no clear understanding of the molecin the fungus Neurospora crassa. J. Bacteriol. 132:511-519. ular nature of any uptake process, but the J., and I. H. Segel. 1974. Transinhibition rate-limiting step has traditionally been inter- 2. Cuppoletti. kinetics of the sulfate transport system ofPenicillihun in because of preted terms of protein "carriers" notatum: analysis based on an Iso Uni Uni velocity the analogy between uptake system kinetics equation. J. Membr. Biol. 17:239-252. and those of many enzymes. Despite its limita- 3. Letham, I). S. 1969. Influence of fertilizer treatment on apple fruit composition and physiology. II. Influtions. this type of interpretation provides a ence on respiration rate and contents of nitrogen, useful basis for considering the control mechaphosphorus. and titratable acidity. Aust. J. Agric. nisms that might be responsible for the changes Res. Z0:1073-1085. in the activities of the uptake systems. Lowen- 4. Lowendorf, H. S., G. F. Bazinet, and C. W. Slayman. 1975. Phosphate transport in Neurospora. Derepresdorf et al. (4) suggested that the high-affinity sion of a high-affinity transport system during phossystem is derepressible because cycloheximide phorus starvation. Biochim. Biophys. Acta 389:541prevented increases in its activity when myce549. lium was transferred to zero phosphate. Our 5. Lowendorf, H. S., C. L. Slayman, and C. W. Slayman. 1974. Phosphate transport in Neurospora. Kinetic finding that Vm,f(HA,, but not Km(HA), is subject characterization of a constitutive, low-affinity transto change depending on the growth conditions port system. Biochim. Biophys. Acta 373:369-382. is consistent with this suggestion, although our 6. Lowendorf, H. S., and C. W. Slayman. 1975. Genetic inability to eliminate the high-affinity system regulation of phosphate transport system II in Neurospora. Biochim. Biophys. Acta 413:95-103. (Fig. 7) indicates that the system has some M. L. 1971. Amino acid transport in Neurospora features of a constitutive system. The simplest 7. Pall, crassa. IV. Properties and regulation of a methionine explanation for the observed changes is that transport system. Biochim. Biophys. Acta 233:201more high-affinity "carriers" of the existing 214.
Vol. 132, No. 2 Printed in U.S.A.
Adaptive Changes in Phosphate Uptake by the Fungus Neurospora crassa in Response to Phosphate Supply ROSS E. BEEVER* AND DONALD J. W. BURNS Plant Diseases Diision, Departmett of Scientific and Industrial Research, Auckland, Newl Zealand Received for publication 17 August 1977
The phosphate uptake rate of Neurospora crassa germlings growing exponentially in media containing phosphate at concentrations between 10 mM and 50 ,uM was virtually constant. The uptake characteristics of these germlings were studied in detail assuming the simultaneous operation of two uptake systems, one of low affinity and one of high affinity. The K,,, of the low-affinity system was constant after growth at phosphate concentrations greater than 1 mM but became progressively lower as the concentration was reduced below 1 mM. In contrast, the K,,, of the high-affinity system was independent of the phosphate concentration of the growth medium. The V,, ,,' of each system was highest after growth at low phosphate concentrations. As the phosphate concentration was increased to a maximum of 100 mM, the V,,,,,,. of the low-afflnity system fell gradually, whereas that of the high-affinity system at first fell rapidly but then reached a constant minimum value at concentrations of 2.5 mM and higher. The differences in the kinetic parameters fully account for the constancy of uptake rate shown by the germlings. There have been many studies of the kinetics of solute uptake systems, and in many instances uptake characteristics have been shown to differ in similar cells which have been exposed to different environments before study. However, there seems to be no instance where the activity of an uptake system(s) in an organism growing under a range of conditions has been precisely related to the requirements of the organism for the solute under those conditions. Such quantitative studies are needed to further our understanding of how uptake systems are controlled. As part of such a study, we have shown that phosphate uptake by Neurospora crassa germlings after growth at either a high (10 mM) or low (50 ,uM) phosphate concentration is due to the simultaneous operation of two uptake systems (1). In the present paper we show that, at the time of study, the growth rate and the rate of phosphate entry of such germlings are independent of the phosphate concentration in the growth medium. The observation made previously (1), that the kinetic parameters of the uptake systems are influenced by the growth phosphate concentration, is extended to germlings growing at various concentrations within the range 50 ,.M to 100 mM. We find that the differences in the kinetic parameters are sufflcient to account precisely for the constancy of phosphate entry during growth. The results permit a detailed description of the way in which N. crassa adapts to phosphate supply.
MATERIALS AND METHOI)S The strain of fungus used and the growth conditions, including the composition of the phosphatefree base medium, have been described (1). For convenience, germlings are referred to by the initial phosphate concentration of the medium in which they were grown (e.g., 50 ,uM germlings were germinated and grown in medium initially containing 50 ,uM phosphate). The slight decreases in concentration that occur during growth have been taken into account where appropriate in calculations and graphs. The time of inoculation is taken as zero time. Estimates of doubling times (t,,) were calculated using least-squares analysis. Where appropriate, measurements are given as the mean + 1 standard deviation. I)ry weight and total cell phosphorus determinations. To determine dry weights, germlings (usually from 500 ml of culture) were harvested onto preweighed cellulose nitrate filters (1.2-,.cm pore size) and washed twice with water. The filter and pad of germlings were dried to constant weight under vacuum over P205. For total cell phosphorus determination, the dried pad and filter were acid-digested. and the phosphate content of the digest was measured (3). Uptake rate measurements with 32p,. Unless otherwise specified, uptake rate measurements were made with our standard method (1), which involves harvesting, washing, and resuspending germlings in fresh medium containing 14 ,M cycloheximide and with 32P, at the chosen concentration. In one experiment (Fig. 4), we also measured uptake with a modified method, which avoids harvesting or washing the germlings and in which cycloheximide is not present in the uptake solution. The procedure
520
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was as follows: at 2 h 20 min, 19 ml of medium containing germlings was transferred from the large growth flask to a smaller flask; at 2 h 26 min, 1 ml of 32Pi-labeled medium of the same phosphate concentration as the growth medium was added. The phosphate uptake rate over the next 8 min was determined from 1-ml samples removed at 1-min intervals. Shaking was continued, and the temperature was maintained at 30°C throughout. Calculation of uptake and estimation of kinetic parameters. It was shown previously (1) that uptake in N. crassa can be accounted for by the operation of two systems, each obeying Michaelis-Menten kinetics. This relationship is expressed by the equation for the double hyperbola:
Vmar HA) S Km(LA) S K",(HA, + S where v is uptake rate; s is phosphate concentration; Vma,LA) and VmaJHA) are the maximum uptake rates of the low- and high-affinity systems, respectively; and K,f(LA) and Km(HA) are the phosphate concentrations that give rise to half-maximum uptake rates for each system. This equation has been used as follows. (i) Calculation of uptake rates: where the values of all four kinetic parameters were known for a given type of germling, the uptake rate at any chosen phosphate concentration was calculated directly. (ii) Estimation of Km values: for a given germling preparation, uptake rates were measured at 15 phosphate concentrations, over a wide concentration range, and a double-hyperbola equation was fitted to the data as described previously (1). (iii) Estimation of V values: the method used to obtain Km values also gives Vnax values, but, because of the large number of uptake measurements required, it was not possible in any one such experiment to measure V nax values for more than two germling types. Vmax values reported in this paper (Fig. 3 and 6) were therefore obtained by using a shorter method that depends on knowing the values for Km(LA) and Km(HA) (Fig. 5). Briefly, for each germling type, uptake rates measured at 1 mM and 10 ,uM phosphate were substituted in turn into the double-hyperbola equation. The resulting two simultaneous equations were then solved for Vmaj.LA) and Vmax,HA) after inserting the Km values. VfnalG8IA) v=--
+
S
+
103
E
102
10
2
0
6 TIME ( h
4
8
10
FIG. 1. Effect of phosphate concentration in the medium on growth of N. crassa. Conidia were inoculated at zero time into flasks containing base medium plus 10 mM (C]), 50 ELM (0), or zero (A) phosphate. Each point represents the harvest from one flask. 80 70 60 0
2
50
X~~
D
40
a 2.
110 0 E E z 30
,;:X
0. -
o m uLU
3:-4
>- I.cc0
13
20
F
J.-
8
IV
3.5
2.5
4.5
TIME ( h)
FIG. 2. Changes in dry weight and total cell phosphorus during growth over 2.5 to 4.5 h. Conidia were inoculated at zero time into flasks of base medium containing 10 mM or 50 /LM phosphate. Dry weight (10 mM germlings [I], td = 2.09 h; 50 .M germlings [0], td = 2.19 h) and total cell P (10 mM germlings [E], td 1.89 h; 50 MM germlings [0], td = 1.81 h) were determined at intervals. Each point represents the mean value of single harvests from two independent experiments. =
RESULTS Growth studies. In a preliminary experiment it was found that increase in dry weight of germlings at a high phosphate concentration (10 mM) became exponential by 2.5 h and time to compare the uptake behavior of germcontinued in exponential fashion at least until lings growing at different phosphate concentra10 h (Fig. 1). If no phosphate was present in tions. At this time exponential growth, as the growth medium, the germling dry weight judged by dry-weight increase, had comfollowed that obtained with the high-phosphate menced, but the phosphate concentration of treatment until about 3 h. If a low concentra- the medium had altered little; for medium tion of phosphate (50 AM) was present, the containing 10 mM phosphate the fall was neggermling dry weight followed that obtained ligible, and for the medium containing 50 ,uM with the high-phosphate treatment until about phosphate the concentration had dropped to 6 h. We chose 2.5 h as the most appropriate about 44 ,M. More detailed study (Fig. 2;
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BEEVER AND BURNS
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unpublished data) confirmed that, over the period 2.5 to 4.5 h, the dry-weight increases of 10 mM and 50 ,uM germlings were the same. By 2.5 h, 50 to 75% of the conidia had produced germ tubes; by 3.5 h over 90% had produced germ tubes. Although the dry weights of the cultures showed an exponential increase from 2.5 h, it does not necessarily follow that all growth parameters were increasing exponentially by this time. We found that although total cell phosphorus at 2.5 h differed slightly between the two types of germlings (Table 1), in each case the rate of its increase per unit volume of culture
01 E
EL
01
was
volume of culture basis so that a direct assessment of the growth-dependent increases in uptake activity for each system could be made. (Elsewhere we have followed the normal practice of expressing V,,,,avalues in terms of tissue dry weight.) Phosphate uptake rates at the concentration used for growth. From knowledge of the total cell phosphorus content at 2.5 h, and the doubling time of this parameter, one can calculate the phosphorus uptake rate of the germlings at 2.5 h. Table 1 shows that estimates of the phosphorus uptake rate for 10 mM and 50 FLM germlings calculated from such data were very similar despite the 200-fold difference in growth phosphate concentration. Because phosphate was the only phosphorus source supplied in the medium and there was no significant phosphorus efflux during uptake (1), we can equate phosphorus uptake measured in this way with phosphate uptake. A less laborious method of determining phosphate uptake at the growth concentration is to measure uptake using 32p;. This was done, by our standard method, for germlings growing at seven phosTABLE 1. Uptake rate estimates based on total cell phosphorus measurements Phosphate
in
Total cell P
germation
at 2.5 h'
medium
cmwIt])
10 mM 50 ,M
558 + 30 507 29
germinationy [dry
Duln paert Doubling Uptake ratet for cell (~tm ol/g [dlry pb (h) wt] per min)
001 25
35 TIME h
45
FIG. 3. Changes in the V ,,,a values (expressed on per-unit-volume of culture basis) of the two phosphate uptake systems during growth ouer 2.5 to 4.5 h. V ,,ax values were calculated from uptake rate a
measurements made at 1 mM and 10 pM phosphate (see text). Symbols: (A) 10 mM germlings-O-, lowaffinity system; *, high-affinity system. (B) 50 ,uM germlings- 0, low-affinity system; 0, high-affinity
system.
E -1
4
-0 E _ 2
CL
10-
PHOSPHATE IN GROWTH MEDIUM
M )
FIG. 4. Effect of phosphate concentration of the growth medium on the phosphate uptake rate. N. crassa germlings were grown at phosphate concentrations from 50 ,M to 10 mM. At 2.5 h, uptake rates at the respective growth phosphate concentrations were measured by either our standard method (a) or the modified method (0). The dotted line is the mean uptake rate (standard method) for all treatments.
tim e
1.89 1.81
3.41 3.24
0.18 0.19
Mean of means of three experiments. on the experiments of Fig. 2. Calculated from the equation: uptake rate = (cell P x ln 2)/doubling time. The standard deviations given are minimum estimates based on the total cell phosphorus measurements only. "
1.0
E
exponential (Fig. 2). The V,/iax values of the uptake systems of both types of germlings were also found to increase in exponential fashion (Fig. 3). In this instance the Vii,ax values have been expressed on a per-unit-
bBased
I
0.01
_
phate concentrations between 10 mM and 50 ,tM (Fig. 4). Uptake rates by this standard procedure were very similar (mean, 3.21 + 0.20 tmol/g [dry weight] per min) irrespective of the phosphate concentration of the growth medium. Use of the modified uptake assay method, which disturbs the germlings less, gave identical results (mean, 3.23 + 0.22 ,umol/ g [dry weight] per min; Fig. 4).
PHOSPHATE UPTAKE IN NEUROSPORA
VOL. 132, 1977
value for 1 mM germlings was close to that obtained for 10 mM germlings, whereas that for 200 ,uM germlings was about midway between the values for 50 ,uM and 1 mM germlings. The K,,,(HA) values of both 200 ,uM and 1 mM germlings were within the range of the values obtained for 50 ,uM and 10 mM germlings. Since the Km HA) values of 10 mM and 50 ,uM germlings were not significantly different (1), we conclude that K,l,HAI is constant (2.75 + 0.34 ,uM, eight determinations) in germlings grown in phosphate concentrations between 50 uM and 10 mM. The Vmar values of 2.5-h germlings grown at seven phosphate concentrations are shown in Fig. 6A. Also shown are the calculated uptake rates, based on these V,,,ar values, for germlings at their respective growth concentrations. Although the calculated rates of uptake were slightly higher at the higher growth concentrations, the mean value of 3.14 + 0.25 ,umol/g (dry weight) per min was close to the mean uptake rate obtained by direct measurement. In an attempt to find whether the high-affinity system could be eliminated completely from germlings, we grew them at very high phosphate concentrations (25 and 100 mM). The uptake rates of these germlings at 10 ,uM and 1 mM phosphate concentrations were close to those of 10 mM germlings. If the assumption is made that Km(LA, and K,,,HA, are the same in 25 mM and 100 mM germlings as in 10 mM germlings, then the Vinax values can be estimated. Values derived in this manner indicate
Differences in the uptake systems after growth at different phosphate concentrations. In the previous paper (1) it was shown that some kinetic parameters of the uptake systems of 10 mM and 50 ,uM germlings were different. In the present paper we have determined K,M, values for germlings growing at 1 mM and 200 pAM phosphate (Fig. 5). The Km(LA) A
1200
0
900
600
-300
u
E I
111
1
,,.
1
B
2~
i0-4
10-3
10-2
PHOSPHATE IN GROWTH MEDIUM ( M )
FIG. 5. Effect of phosphate concentration of the growth medium on Km values of the uptake systems. (A) Low-affinity system; (B) high-affinity system. Each point represents an individual determination of K,1. The solid line through the Km values of the low-affinity system was fitted by inspection. The dotted line through the K,m values of the high-affinity system is the mean of all determinations irrespective of growth concentration. Data for germlings grown at 10 mM and 50 ,uM are from the accompanying paper (1). E
523
B
A
6
Z4
io4
10 -3
o -3 10 -2 PHOSPHATE IN GROWTH MEDIUM (M)
10-2
io -
FIG. 6. Effect of phosphate concentration of the growth medium on V,ax values of the uptake systems. Results from two experiments are shown. (A) Growth phosphate concentrations of 10 mM and below; (B) growth phosphate concentrations of 2.5 mM and above. V,max values were calculated from uptake rate measurements made at 10 IAM and 1 mM phosphate (see text). In (A) Km(HA) was taken as 2.75 tAM and K,m(LA) values were read from Fig. 5. In (B) Km(LA) and Km(HA) were assumed to be the same as for 10 mM germlings. In addition, these kinetic values were used to calculate the uptake rates of the germlings at their respective growth concentrations. The solid lines through the Vmax values have been fitted by inspection; the dotted lines through the calculated uptake values are the means of all values in each experiment. Symbols: 0, Vma,lHA); A, calculated uptake at the growth concentration. V111aZLA);
524
J. BACTERIOL.
BEEVER AND BURNS loo
these small differences in doubling time, it is apparent that growth of the cultures is approximately balanced under the conditions of study. 60 , F Thus, we suggest that results obtained with such germlings can be taken as representative of mycelium at later stages of exponential growth. Changes in the parameters of the uptake O §sWslw Ts ,§TTT ...IT IK systems. The constancy of phosphate uptake 10' rate over a widt concentration range (Table 1, FIG. 7. Diagram showing the relative contribu- Fig. 4) shows that N. crassa exerts a tight control over the activity of its uptake systems. tions of the low- and high-affinity systems to phosphate uptake, at the growth concentration, of 2.5-h Previously, we have established that the kigermlings growing at different phosphate concentra- netic parameters of the uptake systems can tions. Calculated from the data of Fig. 5 and 6A differ depending on the phosphate concentraand B. tion of the growth medium (1). The calculated uptake rates presented in Fig. 6, which are that the high-affinity system does not change based on such kinetic analyses, show that changes in these parameters can account for as the growth phosphate concentration is increased above 10 mM, but the VM,Xr of the low- the observed constancy of uptake. Thus, the affinity system falls (Fig. 6B). The predicted mechanism by which N. crassa adapts to phosuptake rates during growth, calculated using phate supply can be described in detail. At the respective parameter values, were rela- high phosphate concentrations in the growth tively constant over this concentration range medium, uptake is due predominantly to the (mean value, 3.54 + 0.32 ,amol/g [dry weight] low-affinity system [Km(IA,, about 1 mM], as at these concentrations Vm,a,n 1A is so low that the per min). Relative contributions of systems to up- contribution of the high-affinity system is less take. The relative contribution of each uptake than 10% in the total (Fig. 7). Although V,, a. A) system to uptake during growth at a given rises progressively as the growth phosphate phosphate concentration is summarized in Fig. concentrations is lowered from 100 to 1 mM 7. From this diagram it can be predicted that (Fig. 6A and B), this rise is counter-balanced the two systems would contribute equally to by the decreasing phosphate concentration, so uptake in germlings growing at 145 ,tM phos- that the amount contributed by this system to uptake remains relatively constant. At concenphate. trations lower than 1 mM the contribution of DISCUSSION the low-affinity system would decrease rapidly Growth studies. The growth habit of fila- if V,,,aLI,A) and K .,LIA, remained at the values mentous fungi makes them unsuitable for con- found in 1 mM germlings. However, the effectinuous-culture growth techniques. Therefore, tive activity of this system is increased both by a continuing increase in Vm,a,L.A\ to a plateau we chose to use batch culture techniques and study uptake during the period of exponential value at 200 ,uM and below (Fig. 6A), and a growth. Although the dry-weight increase was decrease in K,,,l,1A) (Fig. 5A). Although these exponential by 2.5 h, the dry weight at that changes increase the ability of the low-affinity time was still less than twice that of the inocu- system to mediate phosphate uptake, this syslum (Fig. 1 and 2). We felt it desirable, there- tem alone cannot account for the observed fore, to establish whether the growth parame- uptake rates, and, particularly at the lower ters relating to phosphate uptake were also phosphate concentrations, the high-affinity exponential by this stage. Figures 2 and 3 show system becomes relatively more important. that both the total cell phosphorus and the From a constant low value at phosphate conV,,,,(1 values for both phosphate uptake systems centrations greater than 1 mM, Vi,ar MA) in(expressed on a per-unit of culture basis) in- creases steadily as the concentration in the creased in exponential fashion over the period growth medium decreases, until at 50 tM 2.5 to 4.5 h. The doubling times obtained for VMaxfHA) is over eight times its value at 10 mM these parameters were consistently less than (Fig. 6A). In contrast to the low-affinity systhe doubling time for dry weight. The resultant tem, Km,,(I1A, is unaffected by the external phosslight increase in total cell phosphorus per unit phate concentration (Fig. 5B). of dry weight may reflect developmental In a similar study, also of N. crassa, Lowenchanges during the 2.5- to 4.5-h period. Despite dorf et al. (5. 6) attempted to correlate the v
0
80 _
-
2(20
-
4
z
z
8
40
6
20
_ 8(iO
-
-r
10 4C 2 PHOSPHATE IN GROWTH MEDIUM
"I
M
VOL. 132, 1977
PHOSPHATE UPTAKE IN NEUROSPORA
525
phosphate uptake rate predicted from the activ- type are produced during growth at low phosities of the uptake systems at the phosphate phate concentrations. A similar explanation concentration of the growth medium (37 mM cannot account for the changes in the lowin their case) with the phosphate uptake rate affinity system because K, ,A) as well as as determined from total cell phosphorus and Viazr1LA) can vary. However, such changes can doubling-time values. Using two wild-type be accounted for by models in which the intrastrains, they found that the respective uptake cellular concentration of either the transported rates predicted from kinetic studies were 1.36 substrate or a related metabolite can directly and 1.63 times the rates determined from meas- modify the activity of the uptake system. There urements of total cell phosphorus. These results are a number of precedents for such control of are in contrast to the close agreement we have an uptake system in fungi (2, 7). found. However, the uptake assay used by ACKNOWLED)GNIENT these workers has not been shown to give uptake rates comparable to ones measured Philippa Clark gave excellent technical assistance. without disturbing the fungus. It is possible that their assay procedure. which differs from LITERATURE CITED ours in a number of ways (1), alters the uptake 1. D. J. W., and R. E. Beever. 1977. Kinetic Burns, systems in some respect. characterization of the two phosphate uptake systems There is no clear understanding of the molecin the fungus Neurospora crassa. J. Bacteriol. 132:511-519. ular nature of any uptake process, but the J., and I. H. Segel. 1974. Transinhibition rate-limiting step has traditionally been inter- 2. Cuppoletti. kinetics of the sulfate transport system ofPenicillihun in because of preted terms of protein "carriers" notatum: analysis based on an Iso Uni Uni velocity the analogy between uptake system kinetics equation. J. Membr. Biol. 17:239-252. and those of many enzymes. Despite its limita- 3. Letham, I). S. 1969. Influence of fertilizer treatment on apple fruit composition and physiology. II. Influtions. this type of interpretation provides a ence on respiration rate and contents of nitrogen, useful basis for considering the control mechaphosphorus. and titratable acidity. Aust. J. Agric. nisms that might be responsible for the changes Res. Z0:1073-1085. in the activities of the uptake systems. Lowen- 4. Lowendorf, H. S., G. F. Bazinet, and C. W. Slayman. 1975. Phosphate transport in Neurospora. Derepresdorf et al. (4) suggested that the high-affinity sion of a high-affinity transport system during phossystem is derepressible because cycloheximide phorus starvation. Biochim. Biophys. Acta 389:541prevented increases in its activity when myce549. lium was transferred to zero phosphate. Our 5. Lowendorf, H. S., C. L. Slayman, and C. W. Slayman. 1974. Phosphate transport in Neurospora. Kinetic finding that Vm,f(HA,, but not Km(HA), is subject characterization of a constitutive, low-affinity transto change depending on the growth conditions port system. Biochim. Biophys. Acta 373:369-382. is consistent with this suggestion, although our 6. Lowendorf, H. S., and C. W. Slayman. 1975. Genetic inability to eliminate the high-affinity system regulation of phosphate transport system II in Neurospora. Biochim. Biophys. Acta 413:95-103. (Fig. 7) indicates that the system has some M. L. 1971. Amino acid transport in Neurospora features of a constitutive system. The simplest 7. Pall, crassa. IV. Properties and regulation of a methionine explanation for the observed changes is that transport system. Biochim. Biophys. Acta 233:201more high-affinity "carriers" of the existing 214.
