JOURNAL OF BACTRoILGY, Oct. 1975, p. 232-242 Copyright 0 1975 American Society for Microbiology
Neurospora
crassa
Vol. 124, No. 1 Printed in U.S.A.
Conidial Germination: Role of Endogenous Amino Acid Pools
JOSEPH C. SCHMIT AND STUART BRODY* Department of Biology, University of California at San Diego, La Jolla, California 92037
Received for publication 9 June 1975
The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and y-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.
Many different compounds are efficient ini- tion that these compounds might play in dortiators of the germination of bacterial or fungal mancy and spore germination is not clearly spores (9-11, 24). These include metabolizable understood. This paper reports detailed studies compounds, i.e., amino acids, glucose, inosine, on the changes in the amino acid pools that ocetc., and non-metabolizable compounds, i.e., cur during N. crassa conidial germination. A inorganic salts, dipicolinic acid, furfural, etc. In role is proposed for the degradation of the large addition, many of these same spores can be glutamic acid pool. Other studies on initial induced to germinate by a brief heat shock. biochemical events that occur during conidial After a heat shock, ascospores from Neurospora germination have been reported (5, 22). crassa germinate in deionized water (24). Bacillus megaterium endospores can be induced to MATERIALS AND METHODS germinate in salt solutions by a heat shock (20). N. crassa strain. The wild-type strain of N. crassa, Other spores, such as N. crassa conidia, initiate RL3-8A (FGSC no. was used in these studies some of the metabolic events associated with and can be obtained 2218), from the Fungal Genetics Stock germination as soon as they contact water (14; Center, Humboldt State College, Arcata, Calif. this paper). These results suggest that many Conidial preparation. Conidia were obtained from spores contain endogenous storage compounds agar slant cultures containing 6 ml of Vogel minimal that are used for the initiation of germination. It medium (25) with 2% glucose and 2% agar in test has been proposed that the primary effect of tubes (18 by 150 mm). The cultures were incubated at many of the organic and inorganic initiators 30 C for 2 days and then at 22 C in constant light for additional 5 to 8 days. Forty slants yielded about may be to activate the metabolism of these an 160 mg (dry weight) of conidia. storage compounds (6). A heat shock may have Conidia were harvested with a sterile loop and the same effect. either immediately extracted in ethanol (dry-harHigh levels of readily metabolizable com- vested conidia) or suspended in 100 ml of cold (5 C) pounds such as glutamic acid (8, 16, 22), proline sterile water in a flask containing a magnetic stirring (19, 21), trehalose (2, 12), and polyols (1, 17) bar. In the latter case, the conidial suspension was have been found in dormant spores. The func- vigorously agitated on a magnetic stirrer, and the 232
VOL. 124, 1975
AMINO ACID POOLS
233
contaminating fragments of mycelia were removed by were clarified by centrifugation and stored frozen filtering through four layers of cheesecloth. The coni- until analyzed for free amino acid content. dial preparation was then centrifuged and washed Amino acid analysis. The amino acid content was once with cold, sterile water. The washed conidia were measured on a Beckman 120 C amino acid analyzer either extracted in ethanol (wet-harvested conidia) or equipped with an Infotronics integrator. A summary used immediately as inoculum. The total time re- of the three different procedures that were used is quired for preparing the conidial inoculum was about given in Table 1, and a discussion of the separation 30 min. that was obtained is given in Results. In the experiments with cycloheximide, the inhibiAmino acid standards and other chemicals. The tor (10 gg/ml) was present at all times during the amino acid calibration mixtures and the buffers for preparation of the conidial inoculum. the amino acid analyzer were obtained from Pierce Germination conditions. The standard germina- Chemical Co. Individual amino acids and other comtion medium consisted of 250 ml of Vogel minimal pounds that were used as standards were obtained medium (25) with 2% glucose in 1-liter Erlenmeyer from Sigma Chemical Co. flasks. A nitrate minimal medium, which was used for determining the amount of free amino acids excreted RESULTS into the medium during germination, was the same as Separation of the free amino acids in exthe standard germination medium except that an equimolar amount of nitrate was substituted for tracts of N. crassa. Three different chromatoammonia, and the salts and sugar concentrations graphic procedures employing the amino acid were one-tenth those of the standard medium. When analyzer were used to identify and quantitacycloheximide was used, it was included at 10 jAg/ml tively measure the levels of free amino acids in in the standard germination medium. This concentra- extracts of N. crassa. Procedure A (Table 1) was tion of cycloheximide inhibited germ tube formation the standard two-column procedure designed to completely and inhibited phenylalamine incorporation into hot trichloroacetic acid-insoluble material separate amino acids from hydrolyzed proteins by more than 97% (S. E. Hitchcock, and V. W. (15). This procedure did not separate aspartic Cochrane, Neurospora Newslett. 15:18-19, 1969). In acid from glutathione, serine from the comall cases, germination was initiated by adding freshly bined peak of glutamine plus asparagine, meprepared, wet-harvested conidia to each flask. The thionine from cystathionine, or ornithine from final concentration of conidia was about 5 x 10' lysine. The second chromatographic procedure, spores/ml. All of these cultures were incubated at B (Table 1), was adapted from the conditions 22 C on a rotatory shaker at 125 rpm. for separating amino acids from physiological Harvesting and extraction. Conidia and ger- fluids (Beckman Procedures Manual, Beckman minating conidia were harvested by filtration on membrane filters (EHWP04700, Millipore Corp.). Instruments, Inc.). The only amino acids that Mycelia were harvested on Whatman no. 1 filter were not separated by this procedure were paper. The cells were washed quickly with water and cystine from valine and glutamine from asparathen rapidly plunged into 20 ml of boiling 80% gine. The third chromatographic procedure, C, ethanol. The total time for harvesting was less than 1 was used to measure the glutamine and asparamin. Extracts of dry-harvested conidia were prepared gine levels in dry-harvested conidia. by removing conidia from the slant cultures and Unless otherwise indicated, the levels of the plunging them directly into 20 ml of boiling 80% free amino acids presented in this paper were ethanol. The samples were boiled for 10 min, cooled, obtained using procedure B. All of the samples centrifuged, and filtered through EHWPO4700 mem- were also analyzed using procedure A to meabrane filters (Millipore Corp.). The alcohol-insoluble material was dried overnight at 100 C and weighed to sure the level of free cystine. For those amino determine the residual dry weight (RDW). The super- acids that were cleanly separafed, identical natant, containing the free amino acids, was flash levels were obtained by all three chromatoevaporated to dryness. The dried samples were sus- graphic procedures. The use of three different pended in 3 ml of sodium citrate (pH 2.2) buffer and chromatographic procedures greatly enhanced frozen at -15 C. the reliability of the identification of individual Free amino acids released into the medium amino acids. during germination. After incubating the conidia for Typical chromatographs obtained with procethe desired period of time in either deionized water or dure B of extracts from dry-harvested conidia in nitrate minimal medium, the cells were removed by filtration. Extracts containing the intracellular free are given in Fig. 1. Thirty-six ninhydrin-posiamino acids were prepared as described above. The tive compounds were detected. All of the amino germination medium (water or nitrate minimal me- acids and some of the other ninhydrin-positive dium) was concentrated to dryness with a rotatory compounds were identified by their coflash evaporator. The concentrated medium was dis- chromatography with known standards. Oxisolved in pH 2.2 sodium citrate buffer. The samples dized glutathione was identified as a broad peak
234
SCHMIT AND BRODY
E
r.
e.
E-# = -Z E
zl
L0j
o Q 0
z
Lo
to~
to
c.i
z
$
au
0 4.
toi
LO
e
ss c &. .:S.g* fj
o
.+0
C
+-
0z
z
z
4
u
00
.0
z
Q
00
0.
+
+
aS
0
O
O
_
_
t00
QCOD 0U
Ci
0
E 3-
v
f-
o
o
E
S.
E
s:
O
LAv*k
4
.bC a
LO
+
+
0
-*
cs 4 tz
c-
-
+
+
-I
600 C)
04.
0
o C-
C.
o QA)Q
:~ cli
cm
0 0
to to
co _
..0 at m
c
cm
m
1-
c-1 ci 6 0
LO to
0 Cn
t t
CL) 8.
0o .
-.P 0) O
O
OC
r.
o
0
O
OD
1-X.w -: S-w x: .e U->q.U
-, *wO .
Neurospora
crassa
Vol. 124, No. 1 Printed in U.S.A.
Conidial Germination: Role of Endogenous Amino Acid Pools
JOSEPH C. SCHMIT AND STUART BRODY* Department of Biology, University of California at San Diego, La Jolla, California 92037
Received for publication 9 June 1975
The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and y-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.
Many different compounds are efficient ini- tion that these compounds might play in dortiators of the germination of bacterial or fungal mancy and spore germination is not clearly spores (9-11, 24). These include metabolizable understood. This paper reports detailed studies compounds, i.e., amino acids, glucose, inosine, on the changes in the amino acid pools that ocetc., and non-metabolizable compounds, i.e., cur during N. crassa conidial germination. A inorganic salts, dipicolinic acid, furfural, etc. In role is proposed for the degradation of the large addition, many of these same spores can be glutamic acid pool. Other studies on initial induced to germinate by a brief heat shock. biochemical events that occur during conidial After a heat shock, ascospores from Neurospora germination have been reported (5, 22). crassa germinate in deionized water (24). Bacillus megaterium endospores can be induced to MATERIALS AND METHODS germinate in salt solutions by a heat shock (20). N. crassa strain. The wild-type strain of N. crassa, Other spores, such as N. crassa conidia, initiate RL3-8A (FGSC no. was used in these studies some of the metabolic events associated with and can be obtained 2218), from the Fungal Genetics Stock germination as soon as they contact water (14; Center, Humboldt State College, Arcata, Calif. this paper). These results suggest that many Conidial preparation. Conidia were obtained from spores contain endogenous storage compounds agar slant cultures containing 6 ml of Vogel minimal that are used for the initiation of germination. It medium (25) with 2% glucose and 2% agar in test has been proposed that the primary effect of tubes (18 by 150 mm). The cultures were incubated at many of the organic and inorganic initiators 30 C for 2 days and then at 22 C in constant light for additional 5 to 8 days. Forty slants yielded about may be to activate the metabolism of these an 160 mg (dry weight) of conidia. storage compounds (6). A heat shock may have Conidia were harvested with a sterile loop and the same effect. either immediately extracted in ethanol (dry-harHigh levels of readily metabolizable com- vested conidia) or suspended in 100 ml of cold (5 C) pounds such as glutamic acid (8, 16, 22), proline sterile water in a flask containing a magnetic stirring (19, 21), trehalose (2, 12), and polyols (1, 17) bar. In the latter case, the conidial suspension was have been found in dormant spores. The func- vigorously agitated on a magnetic stirrer, and the 232
VOL. 124, 1975
AMINO ACID POOLS
233
contaminating fragments of mycelia were removed by were clarified by centrifugation and stored frozen filtering through four layers of cheesecloth. The coni- until analyzed for free amino acid content. dial preparation was then centrifuged and washed Amino acid analysis. The amino acid content was once with cold, sterile water. The washed conidia were measured on a Beckman 120 C amino acid analyzer either extracted in ethanol (wet-harvested conidia) or equipped with an Infotronics integrator. A summary used immediately as inoculum. The total time re- of the three different procedures that were used is quired for preparing the conidial inoculum was about given in Table 1, and a discussion of the separation 30 min. that was obtained is given in Results. In the experiments with cycloheximide, the inhibiAmino acid standards and other chemicals. The tor (10 gg/ml) was present at all times during the amino acid calibration mixtures and the buffers for preparation of the conidial inoculum. the amino acid analyzer were obtained from Pierce Germination conditions. The standard germina- Chemical Co. Individual amino acids and other comtion medium consisted of 250 ml of Vogel minimal pounds that were used as standards were obtained medium (25) with 2% glucose in 1-liter Erlenmeyer from Sigma Chemical Co. flasks. A nitrate minimal medium, which was used for determining the amount of free amino acids excreted RESULTS into the medium during germination, was the same as Separation of the free amino acids in exthe standard germination medium except that an equimolar amount of nitrate was substituted for tracts of N. crassa. Three different chromatoammonia, and the salts and sugar concentrations graphic procedures employing the amino acid were one-tenth those of the standard medium. When analyzer were used to identify and quantitacycloheximide was used, it was included at 10 jAg/ml tively measure the levels of free amino acids in in the standard germination medium. This concentra- extracts of N. crassa. Procedure A (Table 1) was tion of cycloheximide inhibited germ tube formation the standard two-column procedure designed to completely and inhibited phenylalamine incorporation into hot trichloroacetic acid-insoluble material separate amino acids from hydrolyzed proteins by more than 97% (S. E. Hitchcock, and V. W. (15). This procedure did not separate aspartic Cochrane, Neurospora Newslett. 15:18-19, 1969). In acid from glutathione, serine from the comall cases, germination was initiated by adding freshly bined peak of glutamine plus asparagine, meprepared, wet-harvested conidia to each flask. The thionine from cystathionine, or ornithine from final concentration of conidia was about 5 x 10' lysine. The second chromatographic procedure, spores/ml. All of these cultures were incubated at B (Table 1), was adapted from the conditions 22 C on a rotatory shaker at 125 rpm. for separating amino acids from physiological Harvesting and extraction. Conidia and ger- fluids (Beckman Procedures Manual, Beckman minating conidia were harvested by filtration on membrane filters (EHWP04700, Millipore Corp.). Instruments, Inc.). The only amino acids that Mycelia were harvested on Whatman no. 1 filter were not separated by this procedure were paper. The cells were washed quickly with water and cystine from valine and glutamine from asparathen rapidly plunged into 20 ml of boiling 80% gine. The third chromatographic procedure, C, ethanol. The total time for harvesting was less than 1 was used to measure the glutamine and asparamin. Extracts of dry-harvested conidia were prepared gine levels in dry-harvested conidia. by removing conidia from the slant cultures and Unless otherwise indicated, the levels of the plunging them directly into 20 ml of boiling 80% free amino acids presented in this paper were ethanol. The samples were boiled for 10 min, cooled, obtained using procedure B. All of the samples centrifuged, and filtered through EHWPO4700 mem- were also analyzed using procedure A to meabrane filters (Millipore Corp.). The alcohol-insoluble material was dried overnight at 100 C and weighed to sure the level of free cystine. For those amino determine the residual dry weight (RDW). The super- acids that were cleanly separafed, identical natant, containing the free amino acids, was flash levels were obtained by all three chromatoevaporated to dryness. The dried samples were sus- graphic procedures. The use of three different pended in 3 ml of sodium citrate (pH 2.2) buffer and chromatographic procedures greatly enhanced frozen at -15 C. the reliability of the identification of individual Free amino acids released into the medium amino acids. during germination. After incubating the conidia for Typical chromatographs obtained with procethe desired period of time in either deionized water or dure B of extracts from dry-harvested conidia in nitrate minimal medium, the cells were removed by filtration. Extracts containing the intracellular free are given in Fig. 1. Thirty-six ninhydrin-posiamino acids were prepared as described above. The tive compounds were detected. All of the amino germination medium (water or nitrate minimal me- acids and some of the other ninhydrin-positive dium) was concentrated to dryness with a rotatory compounds were identified by their coflash evaporator. The concentrated medium was dis- chromatography with known standards. Oxisolved in pH 2.2 sodium citrate buffer. The samples dized glutathione was identified as a broad peak
234
SCHMIT AND BRODY
E
r.
e.
E-# = -Z E
zl
L0j
o Q 0
z
Lo
to~
to
c.i
z
$
au
0 4.
toi
LO
e
ss c &. .:S.g* fj
o
.+0
C
+-
0z
z
z
4
u
00
.0
z
Q
00
0.
+
+
aS
0
O
O
_
_
t00
QCOD 0U
Ci
0
E 3-
v
f-
o
o
E
S.
E
s:
O
LAv*k
4
.bC a
LO
+
+
0
-*
cs 4 tz
c-
-
+
+
-I
600 C)
04.
0
o C-
C.
o QA)Q
:~ cli
cm
0 0
to to
co _
..0 at m
c
cm
m
1-
c-1 ci 6 0
LO to
0 Cn
t t
CL) 8.
0o .
-.P 0) O
O
OC
r.
o
0
O
OD
1-X.w -: S-w x: .e U->q.U
-, *wO .
