DEVELOPMENTAL
BIOLOGY
70, 82-88 (1979)
Genetic Control of Transport Loss during Development Aspergillus nidulans
of
MYRABERMANKURTZANDSEWELL P. CHAMPE Waksman Institute
of Microbiology,
Rutgers-The
State University,
Piscataway,
New Jersey 08854
Received August 23, 1978; accepted in revised form December 14, 1978 The transport of glucose by spore-originated liquid cultures of Aspergillus nidulans varied with culture age. At early times after conidial inoculation, the uptake rate increases, reaches a maximum at about 11 hr, and subsequently declines exponentially. This decline in uptake rate with age is also observed for sucrose, fructose, alanine, and the nonmetabolizable glucose analog, 2-deoxyglucase. Conidiation of liquid-grown Aspergillus nidulans can be induced (by transfer to solid medium) only after a certain developmental stage, called competence, is attained. Two mutants, selected for precocious conidiation on solid medium, differ from wild-type and from each other in the rate of decline of glucose uptake with culture age: The rate of decline is inversely related to the time of conidiation. The precocious development of these mutants is due to a premature acquisition of competence rather than an acceleration of the events that follow induction. We postulate that an internal clock controls the time of acquisition of developmental competence and suggest that this clock is related to changes in a membrane transport system. INTRODUCTION
In submerged liquid culture Aspergillus nidulans will grow indefinitely as undifferentiated hyphae as long as sufficient nutrient levels are maintained. On a nutrient agar surface, however, spore-originated colonies develop differentiated aerial hyphae (conidiophores) which by 24 hr (at 37°C) are distinguishable from ordinary aerial hyphae by their thickened walls and bulbous tips (vesicles). Subsequently, chains of asexual spores (conidia) emerge from the surface of the vesicle thus completing the asexual cycle. Axehod et al. (1973) showed that sporeoriginated liquid-grown colonies can be induced to conidiate by transfer to a solid nutrient surface. If transfer was made at any time before 20 hr, conidiation, as measured by the formation of conidiophore vesicles, always commenced at 24 hr. If, however, transfer was made after 20 hr, conidiation commenced after a constant period of 4 hr. These resuhs were interpreted to mean that a spore-originated colony becomes sensitive to induction at 20 hr. Once 82 00121606/79/05OO82-07$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction m an\ form
Inc. reserved
this sensitivity is acquired, an inducing stimulus results in visible differentiation after a 4-l-u maturation period. This acquired sensitivity to induction was called competence by analogy to similar changes of state exhibited by differentiating cells of higher organisms. Two general models for the acquisition of competence can be conceived: Competence could be a response to a change in the external environment produced by the growth of the organism; for example, depletion of a nutrient or secretion of a compound into the medium. Alternatively, the acquisition of competence could be internally programmed and largely independent of the external environment. This latter view is supported by the liquid-to-solid transfer experiments of Axelrod et al. (1973) in which the inocula densities were very low (l- 10 conidia/ml) and the environment was, thus, effectively constant. On the other hand, the fact that at much higher densities (10:’ to 10” conidia/ml) the time of competence is shortened (Axelrod, personal communication) suggests at least some en-
KURTZ AND CHAMPE
vironmental influence. Nevertheless, it has been shown that, for isolated surface-grown colonies, neither the concentration of a limiting nutrient (glucose) nor continuous replacement of the culture medium has any effect on the time of conidiation and, by inference, on the time of competence (Pastushok and Axelrod, 1976). These observations suggest that the time of competence of a colony is determined by an internal clock that measures development from the time of spore germination. The density effect could be a secondary external influence that accelerates the rate of the clock. In this communication we show that the rate of glucose uptake by wild-type Aspergillus nidulans decreases exponentially from early times after conidial germination. We suggest that this continuous change in uptake rate, or a related parameter, is a possible means by which a colony could measure time. In support of this view we also show that mutations which alter the time of competence also alter the rate of decline of uptake with culture age. MATERIALS
AND
METHODS
Strains. Aspergillus nidulans, wild type (Fungal Genetics Stock Collection No. 4), and the precociously conidiating mutant (BB142) derived from it (Axelrod et al., 1973) were kindly provided by David Axelrod. Strain BP3 was derived from BB142 after ultraviolet mutagenesis using the method of Axelrod et al. (1973). Mycelial growth in liquid culture. Conidia were inoculated at a final concentration of 10”/ml into Cove’s nitrate-less minimal medium as modified by Gealt and Axehod (1974) with 1% glucose as carbon source and 0.01 M glutamate as nitrogen source. Two-liter culture flasks, not more than one-half full, were incubated at 37°C on a rotary shaker at 250 rpm. Under these cultural conditions mycelial colonies aggregate into spherical pellets which become progressively larger and denser with culture age. The yield of a 24-hr culture is about 0.3 mg mycelial dry weight/ml.
Transport
and Development
83
Uptake measurements. Mycelial pellets were harvested by filtration through Miracloth (Chicopee Mills). After washing three times with distilled water, the mycelia were resuspended at a concentration of 0.2 to 0.5 mg dry weight/ml in 100 ml sodium phosphate buffer (0.05 it4, pH 7.0). The mycelia of one-half of the sample were transferred to a weighed filter and dried at 80°C for dry weight measurements; the remaining half was shaken for 30 min in a 250~ml flask at 37°C to deplete residual nutrients. Carbon14-labeled substrate was added to a final concentration of 1.0 m&f (spec. act., 0.01 Ci/mole), and 2.0-ml samples were removed at intervals, filtered immediately through membrane filters (Millipore, 0.45~,amporosity) and washed with 15 ml of water. After drying, the filters were assayed for carbon14 in a Nuclear-Chicago gas flow counter. The slope of the straight line resulting from a plot of the carbon-14 retained versus time is defined as the uptake rate and is expressed as counts per minute of carbon-14 per milligram dry weight per 20 min. In some experiments pellet formation was suppressed either by addition of Carbopol (carboxypolymethylene, B. F. Goodrich Co.) to the minimal medium or by using yeast extract (0.5%)-glucose (2%) medium under otherwise identical cultural conditions. When Carbopol was used it was added to a final concentration of 0.3% and the pH of the medium was readjusted before autoclaving. Nitex filters (No. HD3-52, Tetko, Elmsford, N. Y.) were used to harvest Carbopol-grown cultures since the additive causes clogging of Miracloth. Hexokinase assays. Hexokinase was measured by the method of Roberts (1970) with [‘4C]glucose substituted for [14C]galactose. The reaction mixture (final volume, 1.0 ml) contained: Tris buffer (pH 7.4), 25 pmole; EDTA, 0.5 pmole; MgCle, 2 pmole; ATP, 1 pmole; [‘4C]glucose, 0.2 ,&i, 0.2 pmole (omitted from the control); and cell extract with up to 100 pg protein. After incubation for 15 min at 30°C the reaction was stopped by boiling for 1.5 min, and
a4
DEVELOPMENTAL BIOLOGY VOLUME70,1979
[14C]glucose was added to the control. Denatured protein was removed by centrifugation, and aliquots of the supernatant were spotted on ion exchange filter paper disks (Whatman DE81). After thorough washing, the samples were dried and counted in an automatic gas flow counter. Hexokinase activity is expressed as nanomoles of glucose phosphate per minute per milligram of protein. Time of competence and maturation period. For the measurement of developmental parameters, induction of conidiation was accomplished by the liquid-tosolid transfer technique of Axelrod et al. (1973) at conidial densities less than lo/ml using a medium composed of 0.5% yeast extract plus 2.0% glucose solidified with 1.5% agar for plates. The maturation period is defined as the (constant) interval between transfer and initiation of conidiophore vesicle formation when transfer is made after the time of competence. The time of competence is the earliest time (after conidial inoculation) at which the period between transfer and vesicle formation attains a constant value, i.e., becomes equal to the maturation period. RESULTS
The Rate of Glucose Uptake Decreases with Culture Age The uptake of glucose by Aspergillus nidulans has been investigated by Roman0 and Kornberg (1968) for a variety of culture conditions, but we are aware of no studies which examine this parameter as a function of culture age. Figure 1 shows the age variation in rate of glucose uptake for wildtype A. nidulans from the time of conidial inoculation until the end of logarithmic growth. The rate of uptake per milligram dry weight increases during germination until, at 11 hr, at maximum rate some six times the pregerminated rate is attained. Thereafter, the uptake rate decreases exponentially. The decline of uptake rate is manifested whether the rate is expressed
Id 0
I
I
I
1
10
20
30
40
CULTURE
AGE
Id
(hr)
FIG. 1. Change of glucose uptake rate (bottom panel) and hexokinase activity (top panel) with age of spore-originated cultures of Aspergillus nidulans.
per milligram dry weight or per milligram of protein since, under the cultural conditions used, these two quantities have a constant ratio of 0.12 during exponential growth. The total uptake rate (per unit volume of culture) increases by about lofold between 11 and 30 hr. The decline in uptake rate cannot be attributed to an overall metabolic shutdown since the level of hexokinase activity remains constant throughout the growth period (Fig. 1, top panel), and the growth itself remains exponential. Furthermore, the rate of uptake of the nonmetabolizable glucose analog, 2-deoxyglucose (Brown and Romano, 1969), also declines with age (not shown). In the minimal medium and with the cultural conditions employed in the experiment of Fig. 1, Aspergillus germlings become entangled and grow as multicolonial aggregates (pellets). The pellets are small, loose associations at early times, but later become large and tightly packed. The observed decline in uptake rate could thus be a trivial result of the progressive shielding of the pellet interior. We therefore repeated the uptake measurements in the same medium containing Carbopol, an additive that
KURTZ AND CHAMPE
Transport
prevents pellet formation (Elmayergi et al., 1973), as shown in Fig. 1. It is seen that, although the uptake rate is somewhat higher in the presence of Carbopol, the rate of decline with culture age is the same with or without the additive. We have found that pellet formation by our wild-type strain is also completely suppressed, at least during the first 24 hr of growth, when cultured in yeast extract-glucose liquid medium. Mycelia grown under these conditions, nevertheless, exhibit a decline in glucose uptake at least as rapid as that of mycelial pellets. We conclude that the state of mycelial aggregation cannot explain the decline in uptake rate with age. As seen in Fig. 2 the uptake rates of fructose, sucrose, and alanine also decline exponentially with culture age. Preliminary experiments, however, indicate that the age variation of acetate uptake is complex and qualitatively different from that of glucose.
The Decrease in Uptake Rate with Age Is Not Reversible by Media Replacement or Starvation If the decline of uptake rate with age is I
I
I
10
20
30
due to a change in the medium produced by the growth of the organism, a transfer of mycelial pellets to fresh medium during the period of decline might restore the uptake rate to its maximum value. To test this, pellets from a 24-hr culture were transferred to an equal volume of fresh medium, and the uptake rate was measured after 90 min of further growth. In addition, assays were made on a similarly transferred 24-hr culture which was diluted to the approximate mycelial concentration of a young (11 l-n-) culture. As shown in Table 1, at most, a 30% increase in glucose uptake rate was observed which is far from the value of 380% expected for maximum restoration. Alternatively, it could be argued that the decline in uptake rate may be due to an internal but reversible change, such as accumulation of lipid or carbohydrate. In this case, starvation might be expected to restore the uptake rate to its maximum level. The data given in Table 2 show that starvation of 23-l-u mycelia for as long as 3 hr restores uptake rate by no more than 40% of the unstarved control. The experiments of Tables 1 and 2 thus suggest that the rate
1 ALANINE
FRUCTOSE
0
85
and Development
40
0
IO
CULTURE FIG. 2. Uptake of various metabolites
20
30
40
0
I
I
I
I
IO
20
30
40
AGE (hr)
by Aspergillus
nidulans
during mycelial
growth.
86
DEVELOPMENTAL BIOLOGY TABLE
1
THE EFFECT OF MEDIA REPLACEMENT ON GLUCOSE UPTAKE RATEO Without Transfer Transfer Expected
transfer (undiluted) (diluted) for full restoration
Expt 1
Expt 2
1.00 1.30 1.03 3.80
1.00 0.80 1.30 3.80
n A 24-hr culture was washed by filtration through Miracloth and resuspended in fresh medium either at the same concentration (undiluted) or at a concentration equivalent to an II-hr culture (diluted). After 90 min of growth in the fresh medium, the glucose uptake rate was measured by the procedure described under Materials and Methods (Expt 1) or by the same procedure but with omission of the 30-min starvation period (Expt 2). The values are normalized to the uptake rate of the cultures just before transfer. TABLE
2
THE EFFECT OF STARVATION ON GLUCOSE UPTAKE RATE”
Control Starved Starved and fed Expected for full restoration
Expt lb (23 hr)
Expt 2’ (23 hr)
1.09 1.30 1.30 3.80
1.00 1.40 3.80
n Cultures were washed and starved for the indicated times and either assayed for glucose uptake rate immediately or transferred to fresh medium for 90 min and then assayed. The values are normalized to the uptake rate of the cultures just before starvation. “Starved 90 min in Cove’s medium (less glucose and glutamate). ’ Starved 180 min in Cove’s medium (less glucose and glutamate) + 0.01 M NaN03.
VOLUME 70,1979
ration period following induction, and as shown in Fig. 3, both mutants grow in submerged culture at the same rate as wild type. Axelrod et al. (1973) showed that BB142 germinates at the same rate as wild type, and we have found the same for BP3. Figure 4 shows the change in uptake rate for glucose with culture age for these three strains during exponential growth phase after germination is complete. It is seen that the earlier the time of competence the greater the rate of decline of uptake with culture age as summarized in Table 3. Another difference exhibited by the three strains of Fig. 4 is their relative uptake rates at 11 l-n-the approximate time at which the uptake rate is maximum: the earlier the time of competence the higher the maximum uptake rate. Since, as shown in Table 4, the uptake rates of ungerminated conidia do not differ significantly among the strains, a greater maximum uptake rate must be due to a more rapid increase of uptake rate during germination. Thus, precociousness would appear to be correlated with both an accelerated decline (after 11 hr) and an accelerated increase (before 11 hr) of uptake rate.
of glucose uptake can be taken as an index of mycelial age.
Developmentally Precocious Mutants Are Altered in the Rate of Decline of Uptake with Age Axehod et al. (1973) isolated a precociously conidiating mutant (BB142) which was found to become competent for induction of conidiation 2 hr earlier than wild type. Using BB142 as a parental strain, we isolated a further variant (BP3) which becomes competent even earlier than BB142. Once competent, both mutants develop conidiophores with the normal 4-hr matu-
(3
I
5
IO’
0
//
I IO CULTURE
I
I 20
I 30 AGE
I 40
1
(hr)
FIG. 3. Growth rate of liquid culture of wild-type Aspergillus nidulans (0) and two precocious mutants, BB142, (A) and BP3 (0).
KURTZ AND CHAMPE I WILD
1 I TYPE
I
I
1
1
I
I
I 40
0
20
CULTURE of glucose by wild-type
TABLE
Strain
Wild type BB142 BP3
I
I
I
40
0
I
20
40
AGE (hr)
TABLE
3
OF PRECOCIOUS
1
Aspergillus nidulans and two precocious mutants during mycelial
DEVELOPMENTAL AND TRANSPORT CHARACTERISTICS
I
EP3
20
FIG. 4. Uptake growth.
87
Transport and Development
GLUCOSE
MUTANTS
Time of competence (hr)”
Maturation period (hr)”
Rate of decline of u Ph take (hr- 1
Strain
21 19 17.5
4.5 4.5 4.5
0.065 0.089 0.22
Wild type BBI42 BP3
” The time of competence and the maturation period were measured at 37°C. h Slopes of curves in Fig. 4. Slope = Aln (uptake rate)/Atime. DISCUSSION
The experiments described above show that in liquid cultures of wild-type A. nidulans the uptake rate of glucose and several other metabolites varies with culture age. During germination, the rate increases, reaches a maximum at about 11 hr, and subsequently declines exponentially. The decrease in uptake rate is not due to mycelial pellet formation since it is observed under two different growth conditions which suppress pellet formation. It might be argued that, if newly formed hyphal tips are the only cells of the mycelium active in transport, the observed decline in uptake rate could be due to a chang-
UPTAKE
4
RATE OF UNGERMINATED CONIDIA~
Uptake per mil- Uptake per 10’ viable conidia’ ligram dry weight’ 2990 2340 2140
337 299 341
n Approximately l-2 x lo9 freshly harvested conidia in 0.01% Tween 80 were centrifuged, washed once with distilled water, and resuspended in 50 ml of sodium phosphate buffer (0.05 M, pH 7.0). The uptake assay procedure is the same as that for mycelial pellets, except that the labeled substrate was added to give a lo-fold higher specific activity. ‘Counts per minute per 20 min per milligram dry weight. ’ Counts per minute per 20 min per lo7 viable conidia.
ing ratio of tip cells to total mycelial mass. Fiddy and Trinci (1976) have shown, however, that the ratio of hyphal tips to total mycelial length is a constant, at least for surface-grown colonies. Moreover, in other studies (to be reported elsewhere), we have found that the rate of acetate uptake actually increases during the period of 15 to 20 hr when the rate of glucose uptake is in exponential decline. It is thus difficult to
88
DEVELOPMENTALBIOLOGY
explain the decreasing rate of glucose uptake in terms of mycelial geometry or hyphal heterogeneity. The aim of this study was to identify a physiological parameter which might function as a developmental clock. The continuous decline of uptake rate with mycelial age and its irreversibility by medium replacement or starvation suggests that transport shut-off may be endogenously programmed and could thus serve as a timing mechanism. The fact that mutations which accelerate development also accelerate transport shut-off lends suport to this notion. Since we have examined only three strains, it may, of course, be fortuitous that their times of competence vary inversely, with the rate of decline of uptake. Additional precocious mutants as well as developmentally retarded mutants are being sought to test the generality of this inverse relationship. Although the present results imply a coupling between the developmental schedule and transport activity, this is not to say that some particular value of the uptake rate is the signal that initiates development. The scheduling of transport changes and the initiation of development both could be determined by some overall common timing device which is independently coupled to each process. In this case, mutations affecting the specific coupling mechanisms would be expected to alter each process (transport and development) independently. Mutants selected for specific transport defects are being studied in this respect. Studies with a variety of organisms suggest that oscillatory behavior (e.g., circadian rhythms) is, at least in part, determined by components of the plasma membrane, the primary organelle for metabolite transport. A recent study has shown, for example, that a mutant of Neurospora with altered growth periodicity is deficient in a low-affinity glucose transport system (Halaban, 1975). A current molecular model for circadian rhythms proposes that the oscil-
VOLUME 70,1979
latory period is determined by periodic loading and unloading of the membrane with specific transport proteins (Schweiger and Schweiger, 1977). Although the developmental changes exhibited by A. nidulans are not circadian or periodic, our findings suggest that their time of occurrence might also be determined by a membrane-coupled mechanism. We wish to express our special appreciation to David Axelrod for advice, encouragement, and materials. We gratefully acknowledge also the contributions and assistance of Amy Chang, Lawrence Yager, Nancy Butnick, and Nancy E. L. Hall. This investigation was supported in part by Public Health Service Grant GM17020 from the National Institute of General Medical Sciences and the Charles and Johanna Busch Memorial Fund. REFERENCES AXELROD, D. E., GEALT, M., and PASTUSHOK,M. (1973). Gene control of developmental competence in Aspergillus nidulans. Develop. Biol. 34, 9-15. BROWN, C. E., and ROMANO,A. H. (1969). Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans. J. Bacterial. 100, 1198-1203. ELMAYERGI, H., SCHARER,J. M., and MOO-YOUNG, M. (1973). Effects of polymer additives on fermentation parameters in a culture of A. niger. Biotechnol. Bioeng. 15,845-859. FIDDY, C., and TRINCI, A. P. J. (1976). Mitosis, septation, branching and the duplication cycle in Aspergillus
nidulans.
J. Gen. Microbial.
97, 169-164.
GEALT, M. A., and AXELROD,D. E. (1974). Coordinate regulation of enzyme inducibility and developmental competence in Aspergillus nidulans. Develop.Biol. 41,224-232. HALABAN, R. (1975). Glucose transport-deficient mutants of Neurospora crassa with an unusual rhythmic growth pattern. J. Bacterial. 121, 10561063. PASTUSHOK,M., and AXELROD,D. E. (1976). Effect of glucose, ammonium, and media maintenance on the time of conidiophore initiation by surface colonies of Aspergillus nidulans. J. Gen. Microbial. 94,221224. ROBERTS,C. F. (1970) Enzyme lesions in galactose non-utilizing mutants of Aspergillus nidulans. Biochim. Biophys. Acta 201, 267-283. ROMANO,A. H., and KORNBERG,H. L. (1968). Regulation of sugar utilization by Aspergillus nidulans. Biochim. Biophys. Acta 158,491-493. SCHWEIGER,H., and SCHWEIGER,M. (1977). Circadian rhythms in unicellular organisms: an endeavor to explain the molecular mechanism. Int. Reu. Cytol. 51,315-342.
BIOLOGY
70, 82-88 (1979)
Genetic Control of Transport Loss during Development Aspergillus nidulans
of
MYRABERMANKURTZANDSEWELL P. CHAMPE Waksman Institute
of Microbiology,
Rutgers-The
State University,
Piscataway,
New Jersey 08854
Received August 23, 1978; accepted in revised form December 14, 1978 The transport of glucose by spore-originated liquid cultures of Aspergillus nidulans varied with culture age. At early times after conidial inoculation, the uptake rate increases, reaches a maximum at about 11 hr, and subsequently declines exponentially. This decline in uptake rate with age is also observed for sucrose, fructose, alanine, and the nonmetabolizable glucose analog, 2-deoxyglucase. Conidiation of liquid-grown Aspergillus nidulans can be induced (by transfer to solid medium) only after a certain developmental stage, called competence, is attained. Two mutants, selected for precocious conidiation on solid medium, differ from wild-type and from each other in the rate of decline of glucose uptake with culture age: The rate of decline is inversely related to the time of conidiation. The precocious development of these mutants is due to a premature acquisition of competence rather than an acceleration of the events that follow induction. We postulate that an internal clock controls the time of acquisition of developmental competence and suggest that this clock is related to changes in a membrane transport system. INTRODUCTION
In submerged liquid culture Aspergillus nidulans will grow indefinitely as undifferentiated hyphae as long as sufficient nutrient levels are maintained. On a nutrient agar surface, however, spore-originated colonies develop differentiated aerial hyphae (conidiophores) which by 24 hr (at 37°C) are distinguishable from ordinary aerial hyphae by their thickened walls and bulbous tips (vesicles). Subsequently, chains of asexual spores (conidia) emerge from the surface of the vesicle thus completing the asexual cycle. Axehod et al. (1973) showed that sporeoriginated liquid-grown colonies can be induced to conidiate by transfer to a solid nutrient surface. If transfer was made at any time before 20 hr, conidiation, as measured by the formation of conidiophore vesicles, always commenced at 24 hr. If, however, transfer was made after 20 hr, conidiation commenced after a constant period of 4 hr. These resuhs were interpreted to mean that a spore-originated colony becomes sensitive to induction at 20 hr. Once 82 00121606/79/05OO82-07$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction m an\ form
Inc. reserved
this sensitivity is acquired, an inducing stimulus results in visible differentiation after a 4-l-u maturation period. This acquired sensitivity to induction was called competence by analogy to similar changes of state exhibited by differentiating cells of higher organisms. Two general models for the acquisition of competence can be conceived: Competence could be a response to a change in the external environment produced by the growth of the organism; for example, depletion of a nutrient or secretion of a compound into the medium. Alternatively, the acquisition of competence could be internally programmed and largely independent of the external environment. This latter view is supported by the liquid-to-solid transfer experiments of Axelrod et al. (1973) in which the inocula densities were very low (l- 10 conidia/ml) and the environment was, thus, effectively constant. On the other hand, the fact that at much higher densities (10:’ to 10” conidia/ml) the time of competence is shortened (Axelrod, personal communication) suggests at least some en-
KURTZ AND CHAMPE
vironmental influence. Nevertheless, it has been shown that, for isolated surface-grown colonies, neither the concentration of a limiting nutrient (glucose) nor continuous replacement of the culture medium has any effect on the time of conidiation and, by inference, on the time of competence (Pastushok and Axelrod, 1976). These observations suggest that the time of competence of a colony is determined by an internal clock that measures development from the time of spore germination. The density effect could be a secondary external influence that accelerates the rate of the clock. In this communication we show that the rate of glucose uptake by wild-type Aspergillus nidulans decreases exponentially from early times after conidial germination. We suggest that this continuous change in uptake rate, or a related parameter, is a possible means by which a colony could measure time. In support of this view we also show that mutations which alter the time of competence also alter the rate of decline of uptake with culture age. MATERIALS
AND
METHODS
Strains. Aspergillus nidulans, wild type (Fungal Genetics Stock Collection No. 4), and the precociously conidiating mutant (BB142) derived from it (Axelrod et al., 1973) were kindly provided by David Axelrod. Strain BP3 was derived from BB142 after ultraviolet mutagenesis using the method of Axelrod et al. (1973). Mycelial growth in liquid culture. Conidia were inoculated at a final concentration of 10”/ml into Cove’s nitrate-less minimal medium as modified by Gealt and Axehod (1974) with 1% glucose as carbon source and 0.01 M glutamate as nitrogen source. Two-liter culture flasks, not more than one-half full, were incubated at 37°C on a rotary shaker at 250 rpm. Under these cultural conditions mycelial colonies aggregate into spherical pellets which become progressively larger and denser with culture age. The yield of a 24-hr culture is about 0.3 mg mycelial dry weight/ml.
Transport
and Development
83
Uptake measurements. Mycelial pellets were harvested by filtration through Miracloth (Chicopee Mills). After washing three times with distilled water, the mycelia were resuspended at a concentration of 0.2 to 0.5 mg dry weight/ml in 100 ml sodium phosphate buffer (0.05 it4, pH 7.0). The mycelia of one-half of the sample were transferred to a weighed filter and dried at 80°C for dry weight measurements; the remaining half was shaken for 30 min in a 250~ml flask at 37°C to deplete residual nutrients. Carbon14-labeled substrate was added to a final concentration of 1.0 m&f (spec. act., 0.01 Ci/mole), and 2.0-ml samples were removed at intervals, filtered immediately through membrane filters (Millipore, 0.45~,amporosity) and washed with 15 ml of water. After drying, the filters were assayed for carbon14 in a Nuclear-Chicago gas flow counter. The slope of the straight line resulting from a plot of the carbon-14 retained versus time is defined as the uptake rate and is expressed as counts per minute of carbon-14 per milligram dry weight per 20 min. In some experiments pellet formation was suppressed either by addition of Carbopol (carboxypolymethylene, B. F. Goodrich Co.) to the minimal medium or by using yeast extract (0.5%)-glucose (2%) medium under otherwise identical cultural conditions. When Carbopol was used it was added to a final concentration of 0.3% and the pH of the medium was readjusted before autoclaving. Nitex filters (No. HD3-52, Tetko, Elmsford, N. Y.) were used to harvest Carbopol-grown cultures since the additive causes clogging of Miracloth. Hexokinase assays. Hexokinase was measured by the method of Roberts (1970) with [‘4C]glucose substituted for [14C]galactose. The reaction mixture (final volume, 1.0 ml) contained: Tris buffer (pH 7.4), 25 pmole; EDTA, 0.5 pmole; MgCle, 2 pmole; ATP, 1 pmole; [‘4C]glucose, 0.2 ,&i, 0.2 pmole (omitted from the control); and cell extract with up to 100 pg protein. After incubation for 15 min at 30°C the reaction was stopped by boiling for 1.5 min, and
a4
DEVELOPMENTAL BIOLOGY VOLUME70,1979
[14C]glucose was added to the control. Denatured protein was removed by centrifugation, and aliquots of the supernatant were spotted on ion exchange filter paper disks (Whatman DE81). After thorough washing, the samples were dried and counted in an automatic gas flow counter. Hexokinase activity is expressed as nanomoles of glucose phosphate per minute per milligram of protein. Time of competence and maturation period. For the measurement of developmental parameters, induction of conidiation was accomplished by the liquid-tosolid transfer technique of Axelrod et al. (1973) at conidial densities less than lo/ml using a medium composed of 0.5% yeast extract plus 2.0% glucose solidified with 1.5% agar for plates. The maturation period is defined as the (constant) interval between transfer and initiation of conidiophore vesicle formation when transfer is made after the time of competence. The time of competence is the earliest time (after conidial inoculation) at which the period between transfer and vesicle formation attains a constant value, i.e., becomes equal to the maturation period. RESULTS
The Rate of Glucose Uptake Decreases with Culture Age The uptake of glucose by Aspergillus nidulans has been investigated by Roman0 and Kornberg (1968) for a variety of culture conditions, but we are aware of no studies which examine this parameter as a function of culture age. Figure 1 shows the age variation in rate of glucose uptake for wildtype A. nidulans from the time of conidial inoculation until the end of logarithmic growth. The rate of uptake per milligram dry weight increases during germination until, at 11 hr, at maximum rate some six times the pregerminated rate is attained. Thereafter, the uptake rate decreases exponentially. The decline of uptake rate is manifested whether the rate is expressed
Id 0
I
I
I
1
10
20
30
40
CULTURE
AGE
Id
(hr)
FIG. 1. Change of glucose uptake rate (bottom panel) and hexokinase activity (top panel) with age of spore-originated cultures of Aspergillus nidulans.
per milligram dry weight or per milligram of protein since, under the cultural conditions used, these two quantities have a constant ratio of 0.12 during exponential growth. The total uptake rate (per unit volume of culture) increases by about lofold between 11 and 30 hr. The decline in uptake rate cannot be attributed to an overall metabolic shutdown since the level of hexokinase activity remains constant throughout the growth period (Fig. 1, top panel), and the growth itself remains exponential. Furthermore, the rate of uptake of the nonmetabolizable glucose analog, 2-deoxyglucose (Brown and Romano, 1969), also declines with age (not shown). In the minimal medium and with the cultural conditions employed in the experiment of Fig. 1, Aspergillus germlings become entangled and grow as multicolonial aggregates (pellets). The pellets are small, loose associations at early times, but later become large and tightly packed. The observed decline in uptake rate could thus be a trivial result of the progressive shielding of the pellet interior. We therefore repeated the uptake measurements in the same medium containing Carbopol, an additive that
KURTZ AND CHAMPE
Transport
prevents pellet formation (Elmayergi et al., 1973), as shown in Fig. 1. It is seen that, although the uptake rate is somewhat higher in the presence of Carbopol, the rate of decline with culture age is the same with or without the additive. We have found that pellet formation by our wild-type strain is also completely suppressed, at least during the first 24 hr of growth, when cultured in yeast extract-glucose liquid medium. Mycelia grown under these conditions, nevertheless, exhibit a decline in glucose uptake at least as rapid as that of mycelial pellets. We conclude that the state of mycelial aggregation cannot explain the decline in uptake rate with age. As seen in Fig. 2 the uptake rates of fructose, sucrose, and alanine also decline exponentially with culture age. Preliminary experiments, however, indicate that the age variation of acetate uptake is complex and qualitatively different from that of glucose.
The Decrease in Uptake Rate with Age Is Not Reversible by Media Replacement or Starvation If the decline of uptake rate with age is I
I
I
10
20
30
due to a change in the medium produced by the growth of the organism, a transfer of mycelial pellets to fresh medium during the period of decline might restore the uptake rate to its maximum value. To test this, pellets from a 24-hr culture were transferred to an equal volume of fresh medium, and the uptake rate was measured after 90 min of further growth. In addition, assays were made on a similarly transferred 24-hr culture which was diluted to the approximate mycelial concentration of a young (11 l-n-) culture. As shown in Table 1, at most, a 30% increase in glucose uptake rate was observed which is far from the value of 380% expected for maximum restoration. Alternatively, it could be argued that the decline in uptake rate may be due to an internal but reversible change, such as accumulation of lipid or carbohydrate. In this case, starvation might be expected to restore the uptake rate to its maximum level. The data given in Table 2 show that starvation of 23-l-u mycelia for as long as 3 hr restores uptake rate by no more than 40% of the unstarved control. The experiments of Tables 1 and 2 thus suggest that the rate
1 ALANINE
FRUCTOSE
0
85
and Development
40
0
IO
CULTURE FIG. 2. Uptake of various metabolites
20
30
40
0
I
I
I
I
IO
20
30
40
AGE (hr)
by Aspergillus
nidulans
during mycelial
growth.
86
DEVELOPMENTAL BIOLOGY TABLE
1
THE EFFECT OF MEDIA REPLACEMENT ON GLUCOSE UPTAKE RATEO Without Transfer Transfer Expected
transfer (undiluted) (diluted) for full restoration
Expt 1
Expt 2
1.00 1.30 1.03 3.80
1.00 0.80 1.30 3.80
n A 24-hr culture was washed by filtration through Miracloth and resuspended in fresh medium either at the same concentration (undiluted) or at a concentration equivalent to an II-hr culture (diluted). After 90 min of growth in the fresh medium, the glucose uptake rate was measured by the procedure described under Materials and Methods (Expt 1) or by the same procedure but with omission of the 30-min starvation period (Expt 2). The values are normalized to the uptake rate of the cultures just before transfer. TABLE
2
THE EFFECT OF STARVATION ON GLUCOSE UPTAKE RATE”
Control Starved Starved and fed Expected for full restoration
Expt lb (23 hr)
Expt 2’ (23 hr)
1.09 1.30 1.30 3.80
1.00 1.40 3.80
n Cultures were washed and starved for the indicated times and either assayed for glucose uptake rate immediately or transferred to fresh medium for 90 min and then assayed. The values are normalized to the uptake rate of the cultures just before starvation. “Starved 90 min in Cove’s medium (less glucose and glutamate). ’ Starved 180 min in Cove’s medium (less glucose and glutamate) + 0.01 M NaN03.
VOLUME 70,1979
ration period following induction, and as shown in Fig. 3, both mutants grow in submerged culture at the same rate as wild type. Axelrod et al. (1973) showed that BB142 germinates at the same rate as wild type, and we have found the same for BP3. Figure 4 shows the change in uptake rate for glucose with culture age for these three strains during exponential growth phase after germination is complete. It is seen that the earlier the time of competence the greater the rate of decline of uptake with culture age as summarized in Table 3. Another difference exhibited by the three strains of Fig. 4 is their relative uptake rates at 11 l-n-the approximate time at which the uptake rate is maximum: the earlier the time of competence the higher the maximum uptake rate. Since, as shown in Table 4, the uptake rates of ungerminated conidia do not differ significantly among the strains, a greater maximum uptake rate must be due to a more rapid increase of uptake rate during germination. Thus, precociousness would appear to be correlated with both an accelerated decline (after 11 hr) and an accelerated increase (before 11 hr) of uptake rate.
of glucose uptake can be taken as an index of mycelial age.
Developmentally Precocious Mutants Are Altered in the Rate of Decline of Uptake with Age Axehod et al. (1973) isolated a precociously conidiating mutant (BB142) which was found to become competent for induction of conidiation 2 hr earlier than wild type. Using BB142 as a parental strain, we isolated a further variant (BP3) which becomes competent even earlier than BB142. Once competent, both mutants develop conidiophores with the normal 4-hr matu-
(3
I
5
IO’
0
//
I IO CULTURE
I
I 20
I 30 AGE
I 40
1
(hr)
FIG. 3. Growth rate of liquid culture of wild-type Aspergillus nidulans (0) and two precocious mutants, BB142, (A) and BP3 (0).
KURTZ AND CHAMPE I WILD
1 I TYPE
I
I
1
1
I
I
I 40
0
20
CULTURE of glucose by wild-type
TABLE
Strain
Wild type BB142 BP3
I
I
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40
0
I
20
40
AGE (hr)
TABLE
3
OF PRECOCIOUS
1
Aspergillus nidulans and two precocious mutants during mycelial
DEVELOPMENTAL AND TRANSPORT CHARACTERISTICS
I
EP3
20
FIG. 4. Uptake growth.
87
Transport and Development
GLUCOSE
MUTANTS
Time of competence (hr)”
Maturation period (hr)”
Rate of decline of u Ph take (hr- 1
Strain
21 19 17.5
4.5 4.5 4.5
0.065 0.089 0.22
Wild type BBI42 BP3
” The time of competence and the maturation period were measured at 37°C. h Slopes of curves in Fig. 4. Slope = Aln (uptake rate)/Atime. DISCUSSION
The experiments described above show that in liquid cultures of wild-type A. nidulans the uptake rate of glucose and several other metabolites varies with culture age. During germination, the rate increases, reaches a maximum at about 11 hr, and subsequently declines exponentially. The decrease in uptake rate is not due to mycelial pellet formation since it is observed under two different growth conditions which suppress pellet formation. It might be argued that, if newly formed hyphal tips are the only cells of the mycelium active in transport, the observed decline in uptake rate could be due to a chang-
UPTAKE
4
RATE OF UNGERMINATED CONIDIA~
Uptake per mil- Uptake per 10’ viable conidia’ ligram dry weight’ 2990 2340 2140
337 299 341
n Approximately l-2 x lo9 freshly harvested conidia in 0.01% Tween 80 were centrifuged, washed once with distilled water, and resuspended in 50 ml of sodium phosphate buffer (0.05 M, pH 7.0). The uptake assay procedure is the same as that for mycelial pellets, except that the labeled substrate was added to give a lo-fold higher specific activity. ‘Counts per minute per 20 min per milligram dry weight. ’ Counts per minute per 20 min per lo7 viable conidia.
ing ratio of tip cells to total mycelial mass. Fiddy and Trinci (1976) have shown, however, that the ratio of hyphal tips to total mycelial length is a constant, at least for surface-grown colonies. Moreover, in other studies (to be reported elsewhere), we have found that the rate of acetate uptake actually increases during the period of 15 to 20 hr when the rate of glucose uptake is in exponential decline. It is thus difficult to
88
DEVELOPMENTALBIOLOGY
explain the decreasing rate of glucose uptake in terms of mycelial geometry or hyphal heterogeneity. The aim of this study was to identify a physiological parameter which might function as a developmental clock. The continuous decline of uptake rate with mycelial age and its irreversibility by medium replacement or starvation suggests that transport shut-off may be endogenously programmed and could thus serve as a timing mechanism. The fact that mutations which accelerate development also accelerate transport shut-off lends suport to this notion. Since we have examined only three strains, it may, of course, be fortuitous that their times of competence vary inversely, with the rate of decline of uptake. Additional precocious mutants as well as developmentally retarded mutants are being sought to test the generality of this inverse relationship. Although the present results imply a coupling between the developmental schedule and transport activity, this is not to say that some particular value of the uptake rate is the signal that initiates development. The scheduling of transport changes and the initiation of development both could be determined by some overall common timing device which is independently coupled to each process. In this case, mutations affecting the specific coupling mechanisms would be expected to alter each process (transport and development) independently. Mutants selected for specific transport defects are being studied in this respect. Studies with a variety of organisms suggest that oscillatory behavior (e.g., circadian rhythms) is, at least in part, determined by components of the plasma membrane, the primary organelle for metabolite transport. A recent study has shown, for example, that a mutant of Neurospora with altered growth periodicity is deficient in a low-affinity glucose transport system (Halaban, 1975). A current molecular model for circadian rhythms proposes that the oscil-
VOLUME 70,1979
latory period is determined by periodic loading and unloading of the membrane with specific transport proteins (Schweiger and Schweiger, 1977). Although the developmental changes exhibited by A. nidulans are not circadian or periodic, our findings suggest that their time of occurrence might also be determined by a membrane-coupled mechanism. We wish to express our special appreciation to David Axelrod for advice, encouragement, and materials. We gratefully acknowledge also the contributions and assistance of Amy Chang, Lawrence Yager, Nancy Butnick, and Nancy E. L. Hall. This investigation was supported in part by Public Health Service Grant GM17020 from the National Institute of General Medical Sciences and the Charles and Johanna Busch Memorial Fund. REFERENCES AXELROD, D. E., GEALT, M., and PASTUSHOK,M. (1973). Gene control of developmental competence in Aspergillus nidulans. Develop. Biol. 34, 9-15. BROWN, C. E., and ROMANO,A. H. (1969). Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans. J. Bacterial. 100, 1198-1203. ELMAYERGI, H., SCHARER,J. M., and MOO-YOUNG, M. (1973). Effects of polymer additives on fermentation parameters in a culture of A. niger. Biotechnol. Bioeng. 15,845-859. FIDDY, C., and TRINCI, A. P. J. (1976). Mitosis, septation, branching and the duplication cycle in Aspergillus
nidulans.
J. Gen. Microbial.
97, 169-164.
GEALT, M. A., and AXELROD,D. E. (1974). Coordinate regulation of enzyme inducibility and developmental competence in Aspergillus nidulans. Develop.Biol. 41,224-232. HALABAN, R. (1975). Glucose transport-deficient mutants of Neurospora crassa with an unusual rhythmic growth pattern. J. Bacterial. 121, 10561063. PASTUSHOK,M., and AXELROD,D. E. (1976). Effect of glucose, ammonium, and media maintenance on the time of conidiophore initiation by surface colonies of Aspergillus nidulans. J. Gen. Microbial. 94,221224. ROBERTS,C. F. (1970) Enzyme lesions in galactose non-utilizing mutants of Aspergillus nidulans. Biochim. Biophys. Acta 201, 267-283. ROMANO,A. H., and KORNBERG,H. L. (1968). Regulation of sugar utilization by Aspergillus nidulans. Biochim. Biophys. Acta 158,491-493. SCHWEIGER,H., and SCHWEIGER,M. (1977). Circadian rhythms in unicellular organisms: an endeavor to explain the molecular mechanism. Int. Reu. Cytol. 51,315-342.
