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Studies on the metabolism of acetate by acetate-requiring mutants of Neurospora crassa
Studies on the Metabolism of Acetate by Acetate-Requiring Mutants of Neurospora crussd Bernard S. Strauss Prom the Department
of Zoology,
Syracuse
Received
University,
Syracuse,
Sew I’ork
June 9. 19M
INTRODUCTION
The tricarboxylic acid cycle is generally accept,ed as the major pathway for oxidative carbohydrate metabolism in mammals; no such agreement exists for the microorganisms. Although early doubt#s about the occurrence of the tricarboxylic acid cycle in microorganisms have been dispelled, there is still the feeling that, this cycle may not represent, a major pathway for energy liberat’ion in the bacteria and fungi. Krebs et al. (1) suggest that in Escherichia coli “t,he component reactions of the cycle serve to supply intermediates for organic synt,hesis.” Similarly, Roberts et al. (2) present evidence that in E. coli “The cycle provides more than 50% of the carbon required for protein synthesis but, it is relatively unimportant as a mechanism for oxidat,ion.” An alternative cyclic mechanism, the dicarboxylir acid cycle (Fig. 1) has been proposed by Foster et al. (3) as a mechanism for acetate oxidation in the fungi. The key reaction in this sequence, the formaCon of succinate by direct conjugat’ion of two acetate moieties has never been demonstrated with a purified enzyme system, although Barron and Ghiretti (4) have present’ed some preliminary evidence for its occurrence. In order to st,udy gene interaction at the biochemical level using mutants of Neurospora crassa with lesions in their oxidative carbohydrate metabolism, it was first necessary to define both the usual and potential pathways of acetate oxidation in this organism. Earlier st’udies with “succinir-less” mutants of Neurospora crassa were interpreted by Lewis (5) as demonstrating the existence of a cyclic mechanism for t,he however, as Lewis points conversion of t.he organic acids in Nezcrospora; 1 Supported by contract AT(30-1) I’. S. Atomic Energy Commission.
1138 between 77
Syracuse
‘ITniversity
and the
78
BERNARD S. STRAUSS 1
CH,COOH HOCCOOH
-
-
coon
+-
CO
CW,COOH
G”z
CITRIC ACID ~succinote*mutant block 1
1
TRICARBOXYLIC
ACID
CYCLE
li’ c3
\
f
I lH
c-4 cooli aKETOGLUTARlC AC10
/
itH
-HO%,
-
Ef,,
-
FUMARIC ACID
MALIC ACID
OXALOACETIC ACID
00”
COOH
cool4
COOH
COOH
;;, succ1*1c ACID
COOH PYRUVIC ACID $,
,
HC%H C-H, ACETYLMETHYLCAREINOL (AMCI
+ 0~ block h
DICARBOXYLIC
ACID
CYCLE
c’H3 COOH ACETIC
ACID
adoptive m ‘*wccinOle’ mutants
7
FIG. 1. Scheme of acetate metabolism in Neurospora.
out (5), the data are difficult. to interpret unless it is assumed that the cyclic reactions are mainly synthetic in function. The problem in t,his investigation was t.herefore twofold: What mechanisms arc available fol the oxidation of acetate by Neuroqora, and which of these mechanisms are selected for acetate oxidation in the absence of growth? As a result of these investigations it is now believed that resting cultures of Neurospora oxidize acetat,e by means of a tricarboxylic acid cycle but that a dicarboxylic acid cycle might occur under special conditions. MATERIALS
AND METHODS
The strains of Neurospora crassa used in these studies were the wild-type 7A, the acetate mutant ac-9 sp (6) and the “succinate” mutants 46005 and 464031 (7). Stocks of these strains were kept on slants of minimal medium (8) plus acetate. Starting from an inoculum of coiidia, cultures of 7A and ac-3 sp were grown 2>$ days in 100 ml. of minimal medium plus 0.23 g. NuC2H30?.3H& in 250-ml. Erlenmeyer flasks incubated at 28°C. on a reciprocal shaking machine. The medium was then decanted off, the mycelium washed with pH 6.0, 0.067 M (M/15) phosphate buffer and placed in 100 ml. of buffer. The washed mycclium was then allowed to dissimilate on the shaking machine for the desired period, after which the buffer 2 Stocks of these strains were obtained from Mrs. H. K. Mitchell fornia Institute of Technology.
of the Cali-
METABOLISM OF ACETATE
79
was decanted off and the mycelium transferred to 100 ml. of the test solution in a 25@ml. Erlenmeyer flask. The UC-Ssp mycelium gave a dry weight of about 200 mg. at this point. When the test solution contained Cl4-labeled compounds, a glass-stoppered Erlenmeyer flask was used to which was attached an inverted U -tube containing soda-lime. A vial containing saturated NaOH was suspended above the mycelium to trap respiratory CO* . All experiments were incubated on the shaking machine. At the conclusion of an experiment, the absorbed COS was precipitated as BaC03 and the mycelium was removed by filtering the test solution. In the isotope experiments the mycelium was washed wit,h boiling water several times to remove any absorbed isotope, then dried, weighed, and powdered. The test solution was steam-distilled to separate the accumulated pyruvic acid from acetylmethylcarbinol (AMC, 3-hydroxy-2.butanone) when isolation of derivatives of these compounds was desired; otherwise analyses were made directly on the test solutions. Pyruvate was determined in the residue from the steam distillation by the method of Friedemann and Haugen (Y), AMC in the &earn distillate by the method of Westerfeld (10). A known amount of carricxr diacetyl was added to the steam distillate; the 2,4-dinitrophenylhydrazone of diacetyl \~as then isolated by the method of Green el r/l. (11). A known quantit!of’ carrier pyruvate was added to the residue from the steam distillation, the aolution was made 2 N with respect to HCI, and 0.5$& 2,1-dinitrophenylhydrazinc in 2 S HCl was added. The precipitate of the 2,4-dinitrophenylhydrazone of pyruvic acid was collected, dissolved in 10% Ka&O:I , filtered, and reprecipitated with HCl. It. was t,hen crystallized from ethyl acetate. Acetic acid was determined 1)) titration of an 11-vol. steam distillat,e of medium acidified with HZSOa Citrit acid was det,ermined by t,he method of Ettinger et al. (12). Carhorl-14 assays w(>rr made as described previously (6). The dinitrophenylhydrazones of pyruvic ari(l and diacetyl were plat,ed and counted directly. Hufficicnt counts were recorded to make the probable error of the counting under 5%. RESULTS
Production of Pywic
Acid and AMC’
Pyruvic acid and acetylmethylcarbinol (AMC) accumulate when ac-3 sp is shaken with glucose in buffer (6). Lesser quant’ities of pyruvatc accumulat’e when this strain is shaken with acetate in buffer (Table I). In this latter case AMC accumulation follows a course which has not been explained; the compound could be detected aft’er a short incubation period in buffer or in acetat’e but often disappeared at, t’he conclusion of a 24-hr. shaking period. After 6 hr. incubation, the disappearance of 1.72 mM of acetat’e was associated with the accumulation of 0.014 mJ4 of pyruvate. Although pyruvic acid does accumulate when ac-3 sp mycelium is shaken in buffer with 0.17 Al acet’ate, the addition of acetate reduces the amount, of pyruvic acid accumulat~ed and drastically reduces the
80
BERNARD
S. STRAUSS
I
TABLE Accumulation
of Pyruvic
Acid and Acetylmethylcarbinol ac-3 sp Mycelium
by Xongrowing
Cultures grown 2% days, dissimilated in buffer 16 hr. Glucose l%, in pH 5.9 PO4buffer. Acetate: 2.3 g. NaC?Hs02.3H20 in 100 ml. ‘W/15 KHZPOI . pH = 5.9. All determinations in duplicate. 1ralucs for buffer + acetate average of two sets of flasks run simultaneously. Pymvic
acid,
&jlOO
Contents
Acetylmethylcarbinol,
Time 6.5 _____
Buffer Buffer + glucose Buffer + acetate
ml.
0.45
12.5
0.45
81.0
109.6
14.4
16.4
of incubation, 25
12.5
6.4 75.6 4.4
20.0
ml.
hr.
6.5 ______
1.10 178.1
pY/fOO
0.4 87.1 3.1
25
0.5 163.0 1.G
a In some experiments there is no detectable AMC at this point. amount of AMC accumulated when uc-S sp mycelium is shaken with 1 y. glucose in buffer (Table II). It was first supposed that the metabolism of acetate might serve to decrease the utilization of glucose in a manner analogous to Johnson’s explanation of the Past.eur effect (13); the oxidation of acetate might proceed preferentially and prevent the glycolytic breakdown of glucose by impounding a necessary reactant. Alternatively, some product of acetate metabolism might promote the utilization of both pyruvate and AMC thereby decreasing the amount accumulated. If either of these explanations were correct, the addition of compounds which inhibit acetate metabolism or which prevent the utilization of the energy derived from acetate oxidation by disrupting oxidative phosphorylation would tend to restore the accumulation of pyruvic acid and TABLE II Effect of Acetate on the Accumulation of Pyruvic carbinol by ac-3 sp
Acid and Acetylmethyl-
Cultures grown 2 days. Dissimilated iu buffer 10 hr., incubated 15 hr. Values average of duplicate determinations. Acetic acid added as sodium acet,ate. Acetic 0
Pyruvic acid, p&f/l00 ml. Acetylmethylcarbinol, &f/100 ml.
acid,
mg. 50 mg.
23.6
9.1
18.2
0.5
METABOLISM
TABLE E$ect of Xodium
Fluoroacetate
III
on the Accumulation by ac-3 sp
Cultures grown 2% days, dissimilated in buffer 18 hr. Glucose: 1 g./lOO ml. Acetate: 100 mg. acetic Values as WM accumulated/100 ml. 0
Incubated with
Glucose Glucose
+ acetate
81
OF ACETATE
of Pylrwic
10 hr., incubated acid as sodium
Acid with substrate
acetate/100
ml.
Fluoroacetate concentration, molarity 2.5 X 1OF 5 x 10-t 10’
42.4 23.6
12.8 22.1
16.0 14.4
14.4 18.4
AMC from glucose in the presence of acetate. Incubation of UC-Z sp mycelium with glucose, acetate, and either of the inhibitors sodium fluoroacetate (Table III) or 2,4-dinitrophenol (Table IV) had no such effect. Fluoroacetate itself inhibited the accumulation of pyruvate from glucose by ax-3 sp. Furthermore, incubation of the mutant mycelium with 0.008 M sodium succinate plus glucose increased pyruvate accumulation from 91 p&f/100 ml. to 190 ~&f/100 ml. In order to determine whether acetate increases the utilization of pyruvate by the mutant, both wild-type and ac-3 sp mycelia were incubated with glucose-l-Cl4 in the presence and absence of acetate, and the specific activity of the respiratory CO2 was determined at various time intervals (Table V). If acetate promotes the metabolism of pyruvate by t*he UCsp mutant, then more of the C-l label should appear in the respiratory CO2 when acetate is added since in this mutant the block in carbohydrate metabolism is at t,he stage of pyruvic acid utilization. Actually, acetate depressed the elimination of the C-l carbon of glucose as CO2 in ac-3 sp by 40 y0 after 4 hr., while the wild-type 7A showed only TABLE Effect of Uinitrophenol
IV
on the Accumulation ac-3 sp
of Acefyl,rleth?/lcu,binol
Cultures grown 235 days, dissimilated in buffer 6 hr., incubated 18 hr. Glucose : 1%. Acetate : 150 mg. acetic acid as sodium acetate/l00 Values as PM accumulated/100 ml. Incubated with
Glucose Glucose
plus acetate
Dinitrophenol 0
131. 4.4
with substrate ml.
(sodium salt), pg. 500
87. 4.1
by
82
BERNARD
S. STRAUSS
TABLE Production
V
of CO2 from
Glucose-l-W
Values as counts/min./m&f X 10-d: Glucose 0.5yo; 2.7 X 106 counts/min./mM. Acetate, 100 mg. acetic acid as sodium acetate. Cultures grown 2% days. Dissimilated in buffer 16 hr. Mycelial dry weight at start:
7A = 330mg./flask; UC-~sp = 300 mg./flask. Strain C% trapped in interval
7A GlUCOStZ
Glucose + acetate
a-3
Glucose + fluoroacetate
GlUCOSe
sp
Glucose + acetate
Glucose + fluoroacetate
1.3 1.43 -
2.53 2.92 5.40
hr.
O-4 4-7 7-24
1.21 1.48 2.3
1.0 1.19 1.6
1.37 2.16 2.68
2.17 2.26 2.68
a 17 y. depression. Since nonisotopic CO2 is produced by the simultaneous oxidation of the acetate added, one might expect some dilution of the specific activity of the CO2 produced from glucose. However, there is no apparent reason why there should be a greater reduction of the specific activity of the CO2 produced in the presence of acetate by the mutant than is found in the wild-type control. At any rate, this experiment is evidence against the idea that acetate stimulates the pyruvate metabolism of a&? sp. The higher specific activity of the CO2 produced by the mutant from glucose compared to that produced by wild-type, 7A, may either reflect lower reserves of endogenous substrate in the mutant (which would tend to dilute the CO2 produced from the exogenous isotopic glucose) or it may be due to the greater relative use of the “pentose shunt” by the ac sp strains (6). Pyruvate Accumulation
Before pyruvate formation from acetate could be used as a tool to study acetate metabolism, it was necessary to determine whether factors other than the presence of acetate were involved in pyruvate accumulation. It was particularly important to know whether CO2would be incorporated into pyruvate and to know whether any of the pyruvate accumulated in the presence of acetate came from endogenous sources. The
METABOLISM
OF
TABLE Incorporafion
83
ACETATE
VI
of NaHC’403 into the Accurrlulation from Glucose by ac-3 sp
Cultures grown 236 days, dissimilated (1% glucose) + 2 PC. NaHCY403 24 hr.
in buffer
Products
Produced
16 hr. shaken
with
substrate
Activity
Substance
2.56 X lo5 counts/min./mN 0.282 X lo5 counts/min./mJI 8.1 X 10’ counls/nGn./tng.
Pyruvic acid Acetylmethylcarbinol Mycelium
addition of NaHC1403 to a test solution of glucose in buffer (with the NaOH absorption vial eliminated) led to considerable incorporation of C?” into the pyruvic acid but led to only slight incorporation into the AMC (Table VI). Incorporation of Cl402 into pyruvate has also been noted when C14-labeled bicarbonate is incubated with mycelium and acetate in buffer. To determine whether any of the pyruvate accumulated in the presence of acetate came from the endogenous reserves of the mycelium, cultures of ac-S sp were grown in the presence of glucose-1-U4. After the growth period, t’he medium was decanted off and the mycelium was washed several times with buffer and allowed t’o dissimilate in fresh buffer for 17 hr. The mycelium was washed and then placed in buffer plus unlabeled acetate and incubated for 24 hr. At the conclusion of this period the pyruvate isolated had a specific activity greater than that of the respiratory CO2 (Table VII) indicating that Cl4 did not enter the pyruvate by CO2 fixation and was therefore derived from mycelial constituents by some more direct means. Since no detectable pyruvate is formed by a& sp mycelium when incubated with buffer alone, a test was performed to see whether the TABLE Dual
Origin
of the Pyruvate
VII Accumulated
by ac-3 sp
Labeled acetate = CH&l*OOH, 1.9 X lo5 counts/min./mM; incubation period = 20 hr. Labeled mycelium dissimilated in buffer 17 hr. then shaken in M/15 HH2P0, + 2.3 g. NaC~H30~~3H~0/100 ml. for 24 hr. Fraction
Mycelium, counts/min./mg. Respiratory COZ, counts/min./mM Pyruvic acid, counts/min./mM
Labeled
mycelium
722 1.46 X lo4 4.76 X lo4
Labeled
acetate
165 1.2 x 105 4.0 x 104
84
BERNARD
S. STRAUSS
TABLE
VIII
Eflect of Sodium Ions on the Accumulation
Cultures grown 235 days, dissimilatcd aa above.
of Pyruvic
Acid
in buffer 8 hr., then incubated
Medium
16 hr.
F’yrivic acid accumulated PM
1 g. acetic acid neutralized with KOII in 100 ml. M/15 KHzPOd 1 g. acetic acid neutralized with NaOH in 100 ml. M/15 NaHzPO( 1 g. acetic acid as sodium acetate in 100 ml. M/15 KHtPO, 100 ml. KHZPO, + 0.99 g. NaCl + NaOH to pH 5.9 M/15 phosphate buffer, pH 5.9, KHsPOJN~~IIPO,
17.4 30.5 27.3 4.5 1.2
extra quantity of sodium ion accompanying the acetate was partially responsible for pyruvate accumulat.ion. It had previously been demonstrated (14) that increasing the sodium content of the growth medium leads to pyruvic acid accumulation by wild-t.ype. The data (Table VIII) indicate that a portion of t.he accumulated pyruvate is due to the inTABLE Effect of Sodium
Fluoroacetate
IX
on the Oxidation
of Acetate and Glucose
by xcurospora
Recorded microliters of 02 taken up by a mycclial suspension in the presence of substrate using standard manometric technique. Buffers (final concentrations) 0.034 df tartrate, pH 4.0, 0.067 ,%fphosphate, pH 6.0; fluoroacetatc incubated with mycelium in main vessel during equilibration. Added 20 PM substrate at pH 4.0, 5 PM substrate at pH 6.0. Fluid volume = 3 ml. 7A grown 24 hr. in shake culture on 0.1% sucrose + 0.23 g. NaC~H30?.3H20/100 ml. Collected and washed in 1120 by centrifugation, dissimilated in Fries solution (3) 4 hr., then resuspended in water. UC-Ssp grown 36 hr., dissimilated in pH 6.0 buffer 1 hr., reharvested, minced with scissors, resuspended in buffer and distributed to manometer flasks. Substrate
PH
Time
Concentration of fluoraacetate, molorily 0 2 x 101 4 x 101 1.2 x 10’
Strain
hr.
None Acetatea Glucose5
4.0
2
65 287 221
52 131 63
43 91 79
-
7A
6.0
3
138 229 131
-
-
142 63 94
ac-3 sp
None
Acetatea Glucose0
o Values with acetate and glucose are corrected for the endogenous respiration.
METABOLISM
Growth
TABLE X of Neurospora and Accumulation of Citric of Sodium
85
OF ACETATE
Acid in the
Presence
Fluoroacetate
7A grown 3 days on 100 ml. of medium in shake culture. Acetate when added = 0.23 g. SaCzH302.3H20/100 ml. Dry weights average of two cultures. .4cetate
Dry weight, VI!/.
0 +
Citric acid,” mg./g.
+
0
Fluoroacetate 5 x
368. 398. 0.45
concentration, 10-a 10-x
molarity 4 X 10-3
5 X 1OV
284. 331.
220. 323.
--
20. 232.
-
-
5.12
-
10 2
5. 213. 5.40
dry wt. of mycelizcm
a Analrses for citric acid done on mycelium from separate experiment.
troduction of sodium ions but that over 50 % of the pyruvic acid accumulated is due to the acetate added. Acetate Metabolism The oxidation of acetate by wild-type and ac-3 sp mycelium is easily demonstrable manometrically and there is no obvious comparative difference in the rates of oxidation. In one manomet,ric experiment in which 5 PM of acetate was added, 7A took up an average of 6.6 PM of oxygen while the acetate-requiring mutant ac-3 sp took up 7.8 MM due to the acetate; 10 PM of O2 is required for the complete oxidation of the acetate fed. It has not been possible to demonstrate oxidation of succinate, malat’e, fumarate, or ketoglutarate manometrically using Neurospora mycelium at pH values ranging from pH 3.0 to 6.0. Acetate oxidation is inhibited by sodium fluoroacetate as is glucose oxidation (Table IX). Fluoroacetate also inhibits growth (Table X), and this inhibition is reversed by the addition of acetic acid to the medium. The specific activity of the CO2 obtained from Cl4 carboxyl-labeled acetate (0.17 M) is the same in the presence and absence of fluoroacetate (1F3 M) indicating that fluoroacetate does not inhibit acetate metabolism in the presence of large concentrations of acetate. Cultures of 7A grown in the presence of fluoroacetate accumulate citrate in the mycelium (Table X).I Resting cultures of ac-S sp and 7A accumulate additional quantities of citrate when incubated with acetate in the presence 3 The citrate analyses were performed by Dr. Margaret Wilson of the New York State College of Medicine at Syracuse.
86
BERNARD
S. STRAUSS
TABLE XI Incorporation of C’4H~COOH and CHD400H into the Pyruvic Acid and Acetylmethylcarbinol Accumulated by ac-3 sp Incubated with Acetate
Recorded counts/min./mAf X lo-4except for mycelium where counts/min./mg. given. Acetate = 25 X 10” counts/min./m&f. Cultures grown 2>& days, dissimilated in buffer 8 hr.; incubated in M/15 KHzPOl + 2.3 g. NaCzH302.3Hz0/100 ml. 18 hr. Values averages of two experiments run simultaneously. Substance
Pyruvic acid Acetylmethylcarbinol Respiratory COZ Mycelium, counts/min./mg. QN.S. = count not significantly
CHdYOOH
5.4 N.S.a 10.4 141
C’“HoCOOH
15.9 3.45 7.6 336
above background.
of fluoroacetate. Although fluoroacetate inhibits the oxidation of glucose, the C-l carbon of glucose is preferentially released as COZ in the presence of this inhibitor (Table V). Sodium fluoroacetate probably inhibits that part of glucose oxidation which passes through acetate [or acetyl coenzyme A (&A)] as an intermediate. Since an alternative pathway of glucose oxidation which occurs via the “pentose shunt” liberates the C-l carbon of glucose as CO2 , it is possible that the higher specific activity of the respiratory CO2 produced from glucose in the presence of fluoroacetate is due to the larger relative use of this shunt mechanism in the presence of the inhibitor even though the over-all rate of glucose metabolism is depressed. In order to study the path of acetate oxidation, ac-S sp mycelium was incubated with CY4H&OOH or with CHQ400H (Table XI). If a cyclic mechanism for acetate oxidation were operative, the methyl carbon of acetate would be expected to enter both the pyruvic acid and the AMC while the carboxyl carbon should be found only in the pyruvate (Fig. 1). The methyl carbon of acetate does appear in the AMC, and the pyruvate activity obtained with methyl-labeled acetate is about three times that obtained with carboxyl-labeled acetate. The mycelial activity obtained with methyl-labeled acetate is similarly greater than that obtained with carboxyl-labeled acetate. Studies on the distribution of the carbon atoms of acetate in accumulated products do not permit a distinction to be made between the triand dicarboxylic acid cycles (Fig. 1). However, since pyruvate is an intermediate only in the dicarboxylic acid cycle, the ac sp strains, which
MET.kBOLISY
87
OF ACET.iTE
E d P
oc-jg ---7,
5
15
IO TIME
---.
20
25
(hours)
FIG. 2. Evolution of the methyl of acetate as CO1 . Cultures grown 2;s days, dissimilated in buffer 16 hr., then incubated in M/15 KH2P04 + 0.23 g. SnC~H~0~~3H~O/lOO ml. acetate: WH,COOH: 1.1 X lo6 counts/min./miV. Activity recorded as the average activity of the CO, t,rapped in the time intrrval between the time given and the preceding figure.
are unable to oxidize pyruvic acid at the normal rate (6), should not be able to release the methyl carbon of acetate as CO? as readily as the wild-type if a dicarboxylic acid cycle is operat’ive. T\‘o such effect was found (Fig. 2) ; if anything, ac-3 sp mycelium libera,tes the methyl of acetate as CO? more readily than does the wild-type. (‘Succinate” Mutants The ac mutants require up to 60 mg. of acetate per 20 ml. to produce maximal growth in standing culture (6, 14, 15). When UC-~ (6) is incubated in medium in which xylose has been autoclaved it will grow on the addit,ion of succinate (15). This effect is specific for cc4 (Table XII). It
BERNARD
88
s. STRAYS
TABLE Effect of Succinate
XII
as an Acetate Substitute
Recorded mg. dry wt. after 4 days at 25°C. in 20 ml. of minimal medium plus additions as shown. Autoclaving done for 15 min. at 15 Ib./sq. in. Acetic acid = 2.64 mg./20 ml. as sodium acetate. Succinic acid = 23.8 mg./20 ml. as disodium succinate. -
= I
Xylose + medium auto&wed together
Xylose + medium autoclaved separately No succinate
ac-1 ac-3 W-4
Plus succinate
0 acetate
+ acetate
0 acetate
0
10 Trace 21
0 0 (trace)
0 0 (trace)
No succinate
+ acetate --
0 acetate --
+ acetate
0
13 10 32
6 8 61
0
0 8
Plus succinate 0 acetate ~-
0
0 49
+ acetate
12 12 75
does not seem that the effect of autoclaving is to produce acetate from xylose since the other acetate-requiring strains do not respond to xylose autoclaved with the medium. Up to 40 mg. of succinate per 20 ml. is required to produce maximal response of ac-4 in the presence of autoclaved xylose even when the medium is also supplemented with small quantities of acetate. In contrast to the behavior of the ac mutants, the “succinate” mutants require only about 2.mg. of succinate in 20 ml. of medium to reach maximal dry weight after 4 days in standing culture; they respond to equal concent.rations of malate, fumarate, and a-kctoglutarate (5) (Table TABLE Organic
XIII
Acids as Growth Factors for the “Succinic-less”
Mutant
@JO3
Growth aa mg. dry weight after 4 days at 25°C. in 26 ml. of minimal medium. Supplements neutralized before use. Added 22 +%f of each. Supplement
None Malic acid Fumaric acid Succinic acid a-Ketoglutaric Citric acid Acetic acid 0 Filter sterilized.
Growth
acida
0 76 64 85 61 0 21
METABOLISM OF ACETATE TABLE Response of the “Succinic-less”
XIV Mutant
Growth as mg. dry weight in 20 ml. of minimal acid as sodium acetate. Incubated at 25°C. Growth
period
days 4 5 6 7
89
.@408 to Acetate
medium + 2.5 mg. of acetic
Growth
9.4 22.0 63.6 81.0
XIII). These mutants respond to acetate adaptively (Table XIV) so that maximal growth is produced after 7 days with 2.5 mg. of acetate. The quantitative requirements of these mutants are probably significant since several independent isolations of this type of mutant from various parent strains have similar growth requirements. The ac sp+ strains will also respond adaptively to acetate, but they begin their adaptive response only aft,er a 7-day lag period (6). The UCsp mutants, which do not produce acetate via alternative pathways as readily as do the ac sp+ strains in t’he later stages of t’heir growth cycle, will give maximal growth only on larger amount’s of acetate regardless of the growth period. DISCUSSION
The data are best explained on the assumption that Neurospora normally oxidizes acetate via a tricarboxylic acid cycle, but that a dicarboxylic acid cycle may become operative in certain mutants (Fig. 1). Shemin and Wittenberg (16) have calculated t’he distribution of isotope in tricarboxylic cycle intermediates to be expected after feeding either carboxyl- or methyl-labeled acetate. When these calculat,ions are applied to pyruvic acid it can be shown that the specific activity of the pyruvate obtained from the oxidation of methyl-labeled acetate will bc anywhere from two to five times the activity of the pyruvate obtained from carboxyl-labeled acetate, depending on the number of turns through the cycle taken by the average molecule. The ratio actually obtained (Table XI) was 3: 1 as was the ratio of mycelial activities. Furt,hermore, if acetate oxidation is cyclic, none of the carboxyl carbon of acetate should be found in the AMC, if AMC is formed only from the methyl and carbonyl carbons of pyruvate. None of the carboxyl carbon of acetate is incorporated into the AMC found when preformed ac sp mycelium is shaken
90
BERNARD
S. STRAUSS
with acetate in buffer; the methyl carbon of acetate is incorporated into AMC. As can be seen in Fig. 1, the specific activity of the AMC obtained from methyl-labeled acetate should be greater than the specific activity of the pyruvate; this was not the case. However, while there is evidence that AMC is f&mea from substrate via pyruvate when ac-3 sp is incubated with’glucose (Table I), there is no evidence for a simple precursorproduct relatianship between pyruvic acid and AMC on incubation of UC-Ssp mycelium with acetate, since in this caseAMC appears and then disappears while pyruvic acid is still accumulating. The appearance and subsequent disappearance of AMC on incubation with acetate indicates that some other unknown process occurs, although acetate undoubtedly contributes partially to AMC formation via pyruvate. However, it seems qualitatively significant that only the methyl carbon of acetate enters the AMC, and these results are therefore evidence that acetate is metabolized by a cyclic mechanism. The greater incorporation of NaHCY03 into pyruvic acid than into AMC when UC-S.spmycelium is shaken with glucose and labeled bicarbonate is also consistent with the idea that pyruvate is formed from acetate via a cyclic mechanism. The incorporation of CY402into pyruvic acid can be explained as due to CO2 fixation into oxalacetic acid by combination with pyruvate and randomization of the label by conversion of oxalacetic acid to succinate with a subsequent return to pyruvate. These well-established reaction mechanisms would led to C1402incorporation into the carboxyl of pyruvate and would therefore not lead to incorporation of Ci4 into AMC. If acetate metabolism does occur by a cyclic mechanism in resting cultures, then it is more likely that the cycle is a,tricarboxylic rather than a clicarboxylic acid cycle. Sodium fluoroacetate acts in other organisms by conversion to fluorocitric acid which is an inhibitor of citric acid metabolism (17). The fluoroacetate inhibition of acetate and glucose oxidation by resting cultures of Neurospora and the accumulation of citric acid in the presence of fluoroacetate in both growing and resting cultures are therefore evidence for the utilization of a tricarboxylic acid cycle in Neurospora. Furthermore, the amount of pyruvic acid accumulated by the acetate-requiring mutant when oxidizing acetate is only about 1 y0 of the amount of acetate taken up on a molar basis. Since the ac sp strain used has a nearly complete block in its ability to oxidize pyruvic acid (6), nearly stoichiometric amounts of pyruvic acid would be expected to accumulate if pyruvate were an intermediate in acetate oxidation as required by the dicarboxylic acid cycle. Finally, if pyruvic
METSBOLISM
OF
ACETATE
91
acid were a necessary intermediate in acetate oxidation, then the CK-3sp strain used should be unable to eliminate the methyl of acetate as COZ as readily as the wild-type which has no difficulty in metabolizing pyruvate. Both wild-type and UC-S sp strains do eliminate the methyl of acetate as COZ at equivalent rates and both take up about t’he same amount of oxygen as a result of acetate oxidation at about the same rate. These data therefore eliminate pyruvate as an inbermediate in any mechanism of acetate oxidation by Neurospora. The tricarboxylic acid cycle best fulfils the requirements for a cyclic mechanism of acetate oxidation (to account for the incorporation of t,he methyl carbon of acetate into pyruvate more readily t’han the carboxyl carbon and to account for the failure of the carboxy carbon of acetate to be incorporated into AMC although the methyl carbon is incorporated) which includes citrate as an intermediate (fluoroacetate inhibition) but which does not include pyruvic acid as an intermediat’e. Pyruvate accumulation as a result of acetate oxidation would then be due to “leakage” from the cycle as a result of the breakdown of oxalacetic acid or malic acid. It is not possible to delineate the lesion in the single-gene “succinate” mutants. Failure of a strain of Neurospora to respond to cit,rate (5) is not) necessarily significant since the wild-type itself cannot utilize this compound as a carbon source. It is therefore not possible to decide at present whether the lesion in t)he “succinate” mutants occurs at the level of citrate formation or at the level of the conversion of citrate to Lu-ket)oglutaric acid. Even though these strains do respond adaptively to small quantities of acetate, it is obvious that they are able to make acetate at some stage of their life cycle; strains which do have real difficulty in synthesizing acetate require 10-20 t’imes the quantity of acetate required to give maximal growth of the ‘%uccinat,e” mutants, and the one UCmutant, t’hat responds t’o succinate requires relatively large amounts of this compound. If t)he evidence presented above for the occurrence and use of the tricarboxylic acid cycle as the major route for acetate oxidation by resting cultures of Neurospora is correct, then the “succinate” mutant,s should be unable to use acetate as a growth supplement unless some additional mechanism operates. Either the tricarboxylic acid cycle is blocked only during part of the growth cycle, or some supplementary mechanism for acetate oxidation such as the dicarboxylic acid cycle becomes available upon the addition of acetate or a dicarboxylic acid. It is
92
BERNARD
S. STRAUSS
not possible to decide between these alternatives at present, but either one makes unnecessary the assumption, seemingly required from the growth data, that the “succinate” mutant.s are unable to synthesize acetate. Inhibition of Pyruvic Acid Accumulation Several factors are involved in the interpretation of pyruvic acid accumulation by the acetate-requiring mutants; the sodium-ion content of the medium &ects pyruvate formation from the endogenous reserves of the mycelium (Table VIII), pyruvate is accumulated as a result of the metabolism of glucose or acetate (Table I), CO2 is incorporated into pyruvate (Table VI), and the presence of acet,ate reduces pyruvic acid accumulation from glucose (Table II). The reduction of pyruvic acid accumulation from glucose in the presence of acetate is a true inhibition of pyruvic acid formation by acetate or a closely related substance since (a) inhibitors of acetate metabolism do not restore the accumulation of pyruvate as they should if a metabolic product of acetate were responsible for the effect or if the fact of acetate metabolism alone were enough to depress the accumulation of pyruvate; (b) succinate, which is metabolized via pathways similar to that of acctate, does not reduce pyruvate accumulation; and (c) in the presence of acetate the specific activity of the respiratory CO2 produced from glucase-l-Cl4 is reduced to a greater extent in the ae sp mutant than in the wild-type (Table V). At the very least, this does not support the idea that acetate in some way promotes the metabolism of pyruvate. It is also difficult to understand how the inhibitory effect of fluoroacetate on pyruvic acid accumulation (Table III) is related to its inhibition of acetate metabolism, and this may therefore be a case in which fluoroacetate does not act by prior conversion to fluorocitrate. The phenomenon of the inhibition of precursor accumulation by the substance required for growth has been discussed by Adelberg and Umbarger (18) and by Cohn and Monod (19). Although the mechanisms differ in the various casesanalyzed, this inhibition by feedback seemsto be a method by which the organism can regulate the direction of its metabolic pathways. It does not seem that acetate inhibition of pyruvic acid formation is an inhibition of protein synthesis as suggested by Cohn and Monod (19) for tryptophan and methionine biosynthesis since inhibition is obtained in “resting” cultures.
METABOLISM OF ACETATE
93
ACKNOWLEDGMENTS The aut,hor would like to acknowledge the technical assistance of Mr. George Shaheen, hlr. Joseph Zizzi, and Miss M. L. Jezyk in the course of these investigations.
SUMMARY
1. Resting cultures of an acetate-requiring mutant accumulate pyruvie acid and acetylmethylcarbinol when shaken in buffer with glucose or acet,ate. 2. The methyl carbon of acetate is incorporated into the pyruvate and the acetylmethylcarbinol; the carboxyl carbon enters the pyruvate but not the acetylmethylcarbinol. Neurospora can incorporate COt into pyruvic acid. 3. Sodium fluoroacetate inhibits acetate oxidation and mycelial growth. Fluoroacetate-inhibited cultures accumulate citrate. 4. The met.hyl (barbon of acetate appears as CO2 as readily in acetaterequiring mutants which are unable to oxidize pyruvic acid as in the wild-type. 3. “Succinate” mut’ants Iv-ill grow adaptively on much smaller quantities of acetate t,han required by the ‘(acetate” mutants. 6. It is considered likely that Neurospora ordinarily oxidizes acetate via a t’ricarboxylic acid cycle, but that a dicarboxylic acid cycle might operat’e under certain conditions. 7. Acetate inhibits the accumulation of pyruvate from glucose by acetate-requiring mut)ant.s. REFERENCES 1. KREBS, H. A., GURIN, S., .4x11 EGC~LESTOX, I,. V., Hiochot~. J. 61, 614 (1!152). 2. ROBERTS, R. B., COIVIE, D. B., BRITTEN, R., BELTOX, E., .~NI) ABELSOS, I’. II., Proc. Natl. Acad. Sci. U. S. 39, 1013 (1953). 3. FOSTER, J. W., CARSON, S. F., ANTHONY, D. S., DAVIES, J. B., JEFFERSON, w. E:., ANU Loiw, i% v., Proc. 2C’atl. Acad. sci. I’. 8. 36, 1013 (1949). 4. BARROX, E. S. G., AND GHIRETTI, F., Biochim. et Biophys. Actn 12, 239 (1953). 5. LEWIS, R. W., Am. J. Botany 36, 292 (1948). 6. STRACSS, B. S., AND PIEROG, S., J. Gen. Microbial. 10. 221 (1954). 7. DUBES, GEORGE R., Ph.D. Thesis. California Institute of Technology, June, 1953. 8. BEADLE, G. W., AND TATC’M, E. I,., Am. J. Botany 32, 678 (1945). !I. FRIEDEMAKN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 10. WESTERFELD, W. W., J. Rio/. Chem. 161, 495 (1945).
94
BERNARD
S. STRAUSS
11. GREEN, D. E., WESTERFELD, W. W., VENNESLAND, B., AND KNOX, W. E., J. Bid. Chem. 146, 69 (1942). 12. ETTINGER, R. H., GOLDBAUM, L. R., AND SMITH, L. H., JR., J. Biol. Chem. 199,
531 (1952). 13. JOHNSON, M. T., Science 94, 200 (1941). 14. STRAUSS, B. S., Arch. Biochem. and Biophys. 36, 33 (1952). 15. LEIN, J., APPLEBY, D., AND LEIN, P., Arch. Biochem. and Biophys. 34, 72 (1951). 16. SHEMIN, D., AND WITTENBERG, V., J. Biol. Chem. 192,315 (1951). 17. PETERS, R. A., Proc. Roy. Sot. (London) B139, 143 (1951). 18. ADELBERG, E. A., AND UMBARGER, H. E., J. Biol. Chem. 206, 475 (1953). 19. COHN, M., AND MONOD, J., Sot. Gen. Microbial. Symposia 3, 132 (1953).
of Zoology,
Syracuse
Received
University,
Syracuse,
Sew I’ork
June 9. 19M
INTRODUCTION
The tricarboxylic acid cycle is generally accept,ed as the major pathway for oxidative carbohydrate metabolism in mammals; no such agreement exists for the microorganisms. Although early doubt#s about the occurrence of the tricarboxylic acid cycle in microorganisms have been dispelled, there is still the feeling that, this cycle may not represent, a major pathway for energy liberat’ion in the bacteria and fungi. Krebs et al. (1) suggest that in Escherichia coli “t,he component reactions of the cycle serve to supply intermediates for organic synt,hesis.” Similarly, Roberts et al. (2) present evidence that in E. coli “The cycle provides more than 50% of the carbon required for protein synthesis but, it is relatively unimportant as a mechanism for oxidat,ion.” An alternative cyclic mechanism, the dicarboxylir acid cycle (Fig. 1) has been proposed by Foster et al. (3) as a mechanism for acetate oxidation in the fungi. The key reaction in this sequence, the formaCon of succinate by direct conjugat’ion of two acetate moieties has never been demonstrated with a purified enzyme system, although Barron and Ghiretti (4) have present’ed some preliminary evidence for its occurrence. In order to st,udy gene interaction at the biochemical level using mutants of Neurospora crassa with lesions in their oxidative carbohydrate metabolism, it was first necessary to define both the usual and potential pathways of acetate oxidation in this organism. Earlier st’udies with “succinir-less” mutants of Neurospora crassa were interpreted by Lewis (5) as demonstrating the existence of a cyclic mechanism for t,he however, as Lewis points conversion of t.he organic acids in Nezcrospora; 1 Supported by contract AT(30-1) I’. S. Atomic Energy Commission.
1138 between 77
Syracuse
‘ITniversity
and the
78
BERNARD S. STRAUSS 1
CH,COOH HOCCOOH
-
-
coon
+-
CO
CW,COOH
G”z
CITRIC ACID ~succinote*mutant block 1
1
TRICARBOXYLIC
ACID
CYCLE
li’ c3
\
f
I lH
c-4 cooli aKETOGLUTARlC AC10
/
itH
-HO%,
-
Ef,,
-
FUMARIC ACID
MALIC ACID
OXALOACETIC ACID
00”
COOH
cool4
COOH
COOH
;;, succ1*1c ACID
COOH PYRUVIC ACID $,
,
HC%H C-H, ACETYLMETHYLCAREINOL (AMCI
+ 0~ block h
DICARBOXYLIC
ACID
CYCLE
c’H3 COOH ACETIC
ACID
adoptive m ‘*wccinOle’ mutants
7
FIG. 1. Scheme of acetate metabolism in Neurospora.
out (5), the data are difficult. to interpret unless it is assumed that the cyclic reactions are mainly synthetic in function. The problem in t,his investigation was t.herefore twofold: What mechanisms arc available fol the oxidation of acetate by Neuroqora, and which of these mechanisms are selected for acetate oxidation in the absence of growth? As a result of these investigations it is now believed that resting cultures of Neurospora oxidize acetat,e by means of a tricarboxylic acid cycle but that a dicarboxylic acid cycle might occur under special conditions. MATERIALS
AND METHODS
The strains of Neurospora crassa used in these studies were the wild-type 7A, the acetate mutant ac-9 sp (6) and the “succinate” mutants 46005 and 464031 (7). Stocks of these strains were kept on slants of minimal medium (8) plus acetate. Starting from an inoculum of coiidia, cultures of 7A and ac-3 sp were grown 2>$ days in 100 ml. of minimal medium plus 0.23 g. NuC2H30?.3H& in 250-ml. Erlenmeyer flasks incubated at 28°C. on a reciprocal shaking machine. The medium was then decanted off, the mycelium washed with pH 6.0, 0.067 M (M/15) phosphate buffer and placed in 100 ml. of buffer. The washed mycclium was then allowed to dissimilate on the shaking machine for the desired period, after which the buffer 2 Stocks of these strains were obtained from Mrs. H. K. Mitchell fornia Institute of Technology.
of the Cali-
METABOLISM OF ACETATE
79
was decanted off and the mycelium transferred to 100 ml. of the test solution in a 25@ml. Erlenmeyer flask. The UC-Ssp mycelium gave a dry weight of about 200 mg. at this point. When the test solution contained Cl4-labeled compounds, a glass-stoppered Erlenmeyer flask was used to which was attached an inverted U -tube containing soda-lime. A vial containing saturated NaOH was suspended above the mycelium to trap respiratory CO* . All experiments were incubated on the shaking machine. At the conclusion of an experiment, the absorbed COS was precipitated as BaC03 and the mycelium was removed by filtering the test solution. In the isotope experiments the mycelium was washed wit,h boiling water several times to remove any absorbed isotope, then dried, weighed, and powdered. The test solution was steam-distilled to separate the accumulated pyruvic acid from acetylmethylcarbinol (AMC, 3-hydroxy-2.butanone) when isolation of derivatives of these compounds was desired; otherwise analyses were made directly on the test solutions. Pyruvate was determined in the residue from the steam distillation by the method of Friedemann and Haugen (Y), AMC in the &earn distillate by the method of Westerfeld (10). A known amount of carricxr diacetyl was added to the steam distillate; the 2,4-dinitrophenylhydrazone of diacetyl \~as then isolated by the method of Green el r/l. (11). A known quantit!of’ carrier pyruvate was added to the residue from the steam distillation, the aolution was made 2 N with respect to HCI, and 0.5$& 2,1-dinitrophenylhydrazinc in 2 S HCl was added. The precipitate of the 2,4-dinitrophenylhydrazone of pyruvic acid was collected, dissolved in 10% Ka&O:I , filtered, and reprecipitated with HCl. It. was t,hen crystallized from ethyl acetate. Acetic acid was determined 1)) titration of an 11-vol. steam distillat,e of medium acidified with HZSOa Citrit acid was det,ermined by t,he method of Ettinger et al. (12). Carhorl-14 assays w(>rr made as described previously (6). The dinitrophenylhydrazones of pyruvic ari(l and diacetyl were plat,ed and counted directly. Hufficicnt counts were recorded to make the probable error of the counting under 5%. RESULTS
Production of Pywic
Acid and AMC’
Pyruvic acid and acetylmethylcarbinol (AMC) accumulate when ac-3 sp is shaken with glucose in buffer (6). Lesser quant’ities of pyruvatc accumulat’e when this strain is shaken with acetate in buffer (Table I). In this latter case AMC accumulation follows a course which has not been explained; the compound could be detected aft’er a short incubation period in buffer or in acetat’e but often disappeared at, t’he conclusion of a 24-hr. shaking period. After 6 hr. incubation, the disappearance of 1.72 mM of acetat’e was associated with the accumulation of 0.014 mJ4 of pyruvate. Although pyruvic acid does accumulate when ac-3 sp mycelium is shaken in buffer with 0.17 Al acet’ate, the addition of acetate reduces the amount, of pyruvic acid accumulat~ed and drastically reduces the
80
BERNARD
S. STRAUSS
I
TABLE Accumulation
of Pyruvic
Acid and Acetylmethylcarbinol ac-3 sp Mycelium
by Xongrowing
Cultures grown 2% days, dissimilated in buffer 16 hr. Glucose l%, in pH 5.9 PO4buffer. Acetate: 2.3 g. NaC?Hs02.3H20 in 100 ml. ‘W/15 KHZPOI . pH = 5.9. All determinations in duplicate. 1ralucs for buffer + acetate average of two sets of flasks run simultaneously. Pymvic
acid,
&jlOO
Contents
Acetylmethylcarbinol,
Time 6.5 _____
Buffer Buffer + glucose Buffer + acetate
ml.
0.45
12.5
0.45
81.0
109.6
14.4
16.4
of incubation, 25
12.5
6.4 75.6 4.4
20.0
ml.
hr.
6.5 ______
1.10 178.1
pY/fOO
0.4 87.1 3.1
25
0.5 163.0 1.G
a In some experiments there is no detectable AMC at this point. amount of AMC accumulated when uc-S sp mycelium is shaken with 1 y. glucose in buffer (Table II). It was first supposed that the metabolism of acetate might serve to decrease the utilization of glucose in a manner analogous to Johnson’s explanation of the Past.eur effect (13); the oxidation of acetate might proceed preferentially and prevent the glycolytic breakdown of glucose by impounding a necessary reactant. Alternatively, some product of acetate metabolism might promote the utilization of both pyruvate and AMC thereby decreasing the amount accumulated. If either of these explanations were correct, the addition of compounds which inhibit acetate metabolism or which prevent the utilization of the energy derived from acetate oxidation by disrupting oxidative phosphorylation would tend to restore the accumulation of pyruvic acid and TABLE II Effect of Acetate on the Accumulation of Pyruvic carbinol by ac-3 sp
Acid and Acetylmethyl-
Cultures grown 2 days. Dissimilated iu buffer 10 hr., incubated 15 hr. Values average of duplicate determinations. Acetic acid added as sodium acet,ate. Acetic 0
Pyruvic acid, p&f/l00 ml. Acetylmethylcarbinol, &f/100 ml.
acid,
mg. 50 mg.
23.6
9.1
18.2
0.5
METABOLISM
TABLE E$ect of Xodium
Fluoroacetate
III
on the Accumulation by ac-3 sp
Cultures grown 2% days, dissimilated in buffer 18 hr. Glucose: 1 g./lOO ml. Acetate: 100 mg. acetic Values as WM accumulated/100 ml. 0
Incubated with
Glucose Glucose
+ acetate
81
OF ACETATE
of Pylrwic
10 hr., incubated acid as sodium
Acid with substrate
acetate/100
ml.
Fluoroacetate concentration, molarity 2.5 X 1OF 5 x 10-t 10’
42.4 23.6
12.8 22.1
16.0 14.4
14.4 18.4
AMC from glucose in the presence of acetate. Incubation of UC-Z sp mycelium with glucose, acetate, and either of the inhibitors sodium fluoroacetate (Table III) or 2,4-dinitrophenol (Table IV) had no such effect. Fluoroacetate itself inhibited the accumulation of pyruvate from glucose by ax-3 sp. Furthermore, incubation of the mutant mycelium with 0.008 M sodium succinate plus glucose increased pyruvate accumulation from 91 p&f/100 ml. to 190 ~&f/100 ml. In order to determine whether acetate increases the utilization of pyruvate by the mutant, both wild-type and ac-3 sp mycelia were incubated with glucose-l-Cl4 in the presence and absence of acetate, and the specific activity of the respiratory CO2 was determined at various time intervals (Table V). If acetate promotes the metabolism of pyruvate by t*he UCsp mutant, then more of the C-l label should appear in the respiratory CO2 when acetate is added since in this mutant the block in carbohydrate metabolism is at t,he stage of pyruvic acid utilization. Actually, acetate depressed the elimination of the C-l carbon of glucose as CO2 in ac-3 sp by 40 y0 after 4 hr., while the wild-type 7A showed only TABLE Effect of Uinitrophenol
IV
on the Accumulation ac-3 sp
of Acefyl,rleth?/lcu,binol
Cultures grown 235 days, dissimilated in buffer 6 hr., incubated 18 hr. Glucose : 1%. Acetate : 150 mg. acetic acid as sodium acetate/l00 Values as PM accumulated/100 ml. Incubated with
Glucose Glucose
plus acetate
Dinitrophenol 0
131. 4.4
with substrate ml.
(sodium salt), pg. 500
87. 4.1
by
82
BERNARD
S. STRAUSS
TABLE Production
V
of CO2 from
Glucose-l-W
Values as counts/min./m&f X 10-d: Glucose 0.5yo; 2.7 X 106 counts/min./mM. Acetate, 100 mg. acetic acid as sodium acetate. Cultures grown 2% days. Dissimilated in buffer 16 hr. Mycelial dry weight at start:
7A = 330mg./flask; UC-~sp = 300 mg./flask. Strain C% trapped in interval
7A GlUCOStZ
Glucose + acetate
a-3
Glucose + fluoroacetate
GlUCOSe
sp
Glucose + acetate
Glucose + fluoroacetate
1.3 1.43 -
2.53 2.92 5.40
hr.
O-4 4-7 7-24
1.21 1.48 2.3
1.0 1.19 1.6
1.37 2.16 2.68
2.17 2.26 2.68
a 17 y. depression. Since nonisotopic CO2 is produced by the simultaneous oxidation of the acetate added, one might expect some dilution of the specific activity of the CO2 produced from glucose. However, there is no apparent reason why there should be a greater reduction of the specific activity of the CO2 produced in the presence of acetate by the mutant than is found in the wild-type control. At any rate, this experiment is evidence against the idea that acetate stimulates the pyruvate metabolism of a&? sp. The higher specific activity of the CO2 produced by the mutant from glucose compared to that produced by wild-type, 7A, may either reflect lower reserves of endogenous substrate in the mutant (which would tend to dilute the CO2 produced from the exogenous isotopic glucose) or it may be due to the greater relative use of the “pentose shunt” by the ac sp strains (6). Pyruvate Accumulation
Before pyruvate formation from acetate could be used as a tool to study acetate metabolism, it was necessary to determine whether factors other than the presence of acetate were involved in pyruvate accumulation. It was particularly important to know whether CO2would be incorporated into pyruvate and to know whether any of the pyruvate accumulated in the presence of acetate came from endogenous sources. The
METABOLISM
OF
TABLE Incorporafion
83
ACETATE
VI
of NaHC’403 into the Accurrlulation from Glucose by ac-3 sp
Cultures grown 236 days, dissimilated (1% glucose) + 2 PC. NaHCY403 24 hr.
in buffer
Products
Produced
16 hr. shaken
with
substrate
Activity
Substance
2.56 X lo5 counts/min./mN 0.282 X lo5 counts/min./mJI 8.1 X 10’ counls/nGn./tng.
Pyruvic acid Acetylmethylcarbinol Mycelium
addition of NaHC1403 to a test solution of glucose in buffer (with the NaOH absorption vial eliminated) led to considerable incorporation of C?” into the pyruvic acid but led to only slight incorporation into the AMC (Table VI). Incorporation of Cl402 into pyruvate has also been noted when C14-labeled bicarbonate is incubated with mycelium and acetate in buffer. To determine whether any of the pyruvate accumulated in the presence of acetate came from the endogenous reserves of the mycelium, cultures of ac-S sp were grown in the presence of glucose-1-U4. After the growth period, t’he medium was decanted off and the mycelium was washed several times with buffer and allowed t’o dissimilate in fresh buffer for 17 hr. The mycelium was washed and then placed in buffer plus unlabeled acetate and incubated for 24 hr. At the conclusion of this period the pyruvate isolated had a specific activity greater than that of the respiratory CO2 (Table VII) indicating that Cl4 did not enter the pyruvate by CO2 fixation and was therefore derived from mycelial constituents by some more direct means. Since no detectable pyruvate is formed by a& sp mycelium when incubated with buffer alone, a test was performed to see whether the TABLE Dual
Origin
of the Pyruvate
VII Accumulated
by ac-3 sp
Labeled acetate = CH&l*OOH, 1.9 X lo5 counts/min./mM; incubation period = 20 hr. Labeled mycelium dissimilated in buffer 17 hr. then shaken in M/15 HH2P0, + 2.3 g. NaC~H30~~3H~0/100 ml. for 24 hr. Fraction
Mycelium, counts/min./mg. Respiratory COZ, counts/min./mM Pyruvic acid, counts/min./mM
Labeled
mycelium
722 1.46 X lo4 4.76 X lo4
Labeled
acetate
165 1.2 x 105 4.0 x 104
84
BERNARD
S. STRAUSS
TABLE
VIII
Eflect of Sodium Ions on the Accumulation
Cultures grown 235 days, dissimilatcd aa above.
of Pyruvic
Acid
in buffer 8 hr., then incubated
Medium
16 hr.
F’yrivic acid accumulated PM
1 g. acetic acid neutralized with KOII in 100 ml. M/15 KHzPOd 1 g. acetic acid neutralized with NaOH in 100 ml. M/15 NaHzPO( 1 g. acetic acid as sodium acetate in 100 ml. M/15 KHtPO, 100 ml. KHZPO, + 0.99 g. NaCl + NaOH to pH 5.9 M/15 phosphate buffer, pH 5.9, KHsPOJN~~IIPO,
17.4 30.5 27.3 4.5 1.2
extra quantity of sodium ion accompanying the acetate was partially responsible for pyruvate accumulat.ion. It had previously been demonstrated (14) that increasing the sodium content of the growth medium leads to pyruvic acid accumulation by wild-t.ype. The data (Table VIII) indicate that a portion of t.he accumulated pyruvate is due to the inTABLE Effect of Sodium
Fluoroacetate
IX
on the Oxidation
of Acetate and Glucose
by xcurospora
Recorded microliters of 02 taken up by a mycclial suspension in the presence of substrate using standard manometric technique. Buffers (final concentrations) 0.034 df tartrate, pH 4.0, 0.067 ,%fphosphate, pH 6.0; fluoroacetatc incubated with mycelium in main vessel during equilibration. Added 20 PM substrate at pH 4.0, 5 PM substrate at pH 6.0. Fluid volume = 3 ml. 7A grown 24 hr. in shake culture on 0.1% sucrose + 0.23 g. NaC~H30?.3H20/100 ml. Collected and washed in 1120 by centrifugation, dissimilated in Fries solution (3) 4 hr., then resuspended in water. UC-Ssp grown 36 hr., dissimilated in pH 6.0 buffer 1 hr., reharvested, minced with scissors, resuspended in buffer and distributed to manometer flasks. Substrate
PH
Time
Concentration of fluoraacetate, molorily 0 2 x 101 4 x 101 1.2 x 10’
Strain
hr.
None Acetatea Glucose5
4.0
2
65 287 221
52 131 63
43 91 79
-
7A
6.0
3
138 229 131
-
-
142 63 94
ac-3 sp
None
Acetatea Glucose0
o Values with acetate and glucose are corrected for the endogenous respiration.
METABOLISM
Growth
TABLE X of Neurospora and Accumulation of Citric of Sodium
85
OF ACETATE
Acid in the
Presence
Fluoroacetate
7A grown 3 days on 100 ml. of medium in shake culture. Acetate when added = 0.23 g. SaCzH302.3H20/100 ml. Dry weights average of two cultures. .4cetate
Dry weight, VI!/.
0 +
Citric acid,” mg./g.
+
0
Fluoroacetate 5 x
368. 398. 0.45
concentration, 10-a 10-x
molarity 4 X 10-3
5 X 1OV
284. 331.
220. 323.
--
20. 232.
-
-
5.12
-
10 2
5. 213. 5.40
dry wt. of mycelizcm
a Analrses for citric acid done on mycelium from separate experiment.
troduction of sodium ions but that over 50 % of the pyruvic acid accumulated is due to the acetate added. Acetate Metabolism The oxidation of acetate by wild-type and ac-3 sp mycelium is easily demonstrable manometrically and there is no obvious comparative difference in the rates of oxidation. In one manomet,ric experiment in which 5 PM of acetate was added, 7A took up an average of 6.6 PM of oxygen while the acetate-requiring mutant ac-3 sp took up 7.8 MM due to the acetate; 10 PM of O2 is required for the complete oxidation of the acetate fed. It has not been possible to demonstrate oxidation of succinate, malat’e, fumarate, or ketoglutarate manometrically using Neurospora mycelium at pH values ranging from pH 3.0 to 6.0. Acetate oxidation is inhibited by sodium fluoroacetate as is glucose oxidation (Table IX). Fluoroacetate also inhibits growth (Table X), and this inhibition is reversed by the addition of acetic acid to the medium. The specific activity of the CO2 obtained from Cl4 carboxyl-labeled acetate (0.17 M) is the same in the presence and absence of fluoroacetate (1F3 M) indicating that fluoroacetate does not inhibit acetate metabolism in the presence of large concentrations of acetate. Cultures of 7A grown in the presence of fluoroacetate accumulate citrate in the mycelium (Table X).I Resting cultures of ac-S sp and 7A accumulate additional quantities of citrate when incubated with acetate in the presence 3 The citrate analyses were performed by Dr. Margaret Wilson of the New York State College of Medicine at Syracuse.
86
BERNARD
S. STRAUSS
TABLE XI Incorporation of C’4H~COOH and CHD400H into the Pyruvic Acid and Acetylmethylcarbinol Accumulated by ac-3 sp Incubated with Acetate
Recorded counts/min./mAf X lo-4except for mycelium where counts/min./mg. given. Acetate = 25 X 10” counts/min./m&f. Cultures grown 2>& days, dissimilated in buffer 8 hr.; incubated in M/15 KHzPOl + 2.3 g. NaCzH302.3Hz0/100 ml. 18 hr. Values averages of two experiments run simultaneously. Substance
Pyruvic acid Acetylmethylcarbinol Respiratory COZ Mycelium, counts/min./mg. QN.S. = count not significantly
CHdYOOH
5.4 N.S.a 10.4 141
C’“HoCOOH
15.9 3.45 7.6 336
above background.
of fluoroacetate. Although fluoroacetate inhibits the oxidation of glucose, the C-l carbon of glucose is preferentially released as COZ in the presence of this inhibitor (Table V). Sodium fluoroacetate probably inhibits that part of glucose oxidation which passes through acetate [or acetyl coenzyme A (&A)] as an intermediate. Since an alternative pathway of glucose oxidation which occurs via the “pentose shunt” liberates the C-l carbon of glucose as CO2 , it is possible that the higher specific activity of the respiratory CO2 produced from glucose in the presence of fluoroacetate is due to the larger relative use of this shunt mechanism in the presence of the inhibitor even though the over-all rate of glucose metabolism is depressed. In order to study the path of acetate oxidation, ac-S sp mycelium was incubated with CY4H&OOH or with CHQ400H (Table XI). If a cyclic mechanism for acetate oxidation were operative, the methyl carbon of acetate would be expected to enter both the pyruvic acid and the AMC while the carboxyl carbon should be found only in the pyruvate (Fig. 1). The methyl carbon of acetate does appear in the AMC, and the pyruvate activity obtained with methyl-labeled acetate is about three times that obtained with carboxyl-labeled acetate. The mycelial activity obtained with methyl-labeled acetate is similarly greater than that obtained with carboxyl-labeled acetate. Studies on the distribution of the carbon atoms of acetate in accumulated products do not permit a distinction to be made between the triand dicarboxylic acid cycles (Fig. 1). However, since pyruvate is an intermediate only in the dicarboxylic acid cycle, the ac sp strains, which
MET.kBOLISY
87
OF ACET.iTE
E d P
oc-jg ---7,
5
15
IO TIME
---.
20
25
(hours)
FIG. 2. Evolution of the methyl of acetate as CO1 . Cultures grown 2;s days, dissimilated in buffer 16 hr., then incubated in M/15 KH2P04 + 0.23 g. SnC~H~0~~3H~O/lOO ml. acetate: WH,COOH: 1.1 X lo6 counts/min./miV. Activity recorded as the average activity of the CO, t,rapped in the time intrrval between the time given and the preceding figure.
are unable to oxidize pyruvic acid at the normal rate (6), should not be able to release the methyl carbon of acetate as CO? as readily as the wild-type if a dicarboxylic acid cycle is operat’ive. T\‘o such effect was found (Fig. 2) ; if anything, ac-3 sp mycelium libera,tes the methyl of acetate as CO? more readily than does the wild-type. (‘Succinate” Mutants The ac mutants require up to 60 mg. of acetate per 20 ml. to produce maximal growth in standing culture (6, 14, 15). When UC-~ (6) is incubated in medium in which xylose has been autoclaved it will grow on the addit,ion of succinate (15). This effect is specific for cc4 (Table XII). It
BERNARD
88
s. STRAYS
TABLE Effect of Succinate
XII
as an Acetate Substitute
Recorded mg. dry wt. after 4 days at 25°C. in 20 ml. of minimal medium plus additions as shown. Autoclaving done for 15 min. at 15 Ib./sq. in. Acetic acid = 2.64 mg./20 ml. as sodium acetate. Succinic acid = 23.8 mg./20 ml. as disodium succinate. -
= I
Xylose + medium auto&wed together
Xylose + medium autoclaved separately No succinate
ac-1 ac-3 W-4
Plus succinate
0 acetate
+ acetate
0 acetate
0
10 Trace 21
0 0 (trace)
0 0 (trace)
No succinate
+ acetate --
0 acetate --
+ acetate
0
13 10 32
6 8 61
0
0 8
Plus succinate 0 acetate ~-
0
0 49
+ acetate
12 12 75
does not seem that the effect of autoclaving is to produce acetate from xylose since the other acetate-requiring strains do not respond to xylose autoclaved with the medium. Up to 40 mg. of succinate per 20 ml. is required to produce maximal response of ac-4 in the presence of autoclaved xylose even when the medium is also supplemented with small quantities of acetate. In contrast to the behavior of the ac mutants, the “succinate” mutants require only about 2.mg. of succinate in 20 ml. of medium to reach maximal dry weight after 4 days in standing culture; they respond to equal concent.rations of malate, fumarate, and a-kctoglutarate (5) (Table TABLE Organic
XIII
Acids as Growth Factors for the “Succinic-less”
Mutant
@JO3
Growth aa mg. dry weight after 4 days at 25°C. in 26 ml. of minimal medium. Supplements neutralized before use. Added 22 +%f of each. Supplement
None Malic acid Fumaric acid Succinic acid a-Ketoglutaric Citric acid Acetic acid 0 Filter sterilized.
Growth
acida
0 76 64 85 61 0 21
METABOLISM OF ACETATE TABLE Response of the “Succinic-less”
XIV Mutant
Growth as mg. dry weight in 20 ml. of minimal acid as sodium acetate. Incubated at 25°C. Growth
period
days 4 5 6 7
89
.@408 to Acetate
medium + 2.5 mg. of acetic
Growth
9.4 22.0 63.6 81.0
XIII). These mutants respond to acetate adaptively (Table XIV) so that maximal growth is produced after 7 days with 2.5 mg. of acetate. The quantitative requirements of these mutants are probably significant since several independent isolations of this type of mutant from various parent strains have similar growth requirements. The ac sp+ strains will also respond adaptively to acetate, but they begin their adaptive response only aft,er a 7-day lag period (6). The UCsp mutants, which do not produce acetate via alternative pathways as readily as do the ac sp+ strains in t’he later stages of t’heir growth cycle, will give maximal growth only on larger amount’s of acetate regardless of the growth period. DISCUSSION
The data are best explained on the assumption that Neurospora normally oxidizes acetate via a tricarboxylic acid cycle, but that a dicarboxylic acid cycle may become operative in certain mutants (Fig. 1). Shemin and Wittenberg (16) have calculated t’he distribution of isotope in tricarboxylic cycle intermediates to be expected after feeding either carboxyl- or methyl-labeled acetate. When these calculat,ions are applied to pyruvic acid it can be shown that the specific activity of the pyruvate obtained from the oxidation of methyl-labeled acetate will bc anywhere from two to five times the activity of the pyruvate obtained from carboxyl-labeled acetate, depending on the number of turns through the cycle taken by the average molecule. The ratio actually obtained (Table XI) was 3: 1 as was the ratio of mycelial activities. Furt,hermore, if acetate oxidation is cyclic, none of the carboxyl carbon of acetate should be found in the AMC, if AMC is formed only from the methyl and carbonyl carbons of pyruvate. None of the carboxyl carbon of acetate is incorporated into the AMC found when preformed ac sp mycelium is shaken
90
BERNARD
S. STRAUSS
with acetate in buffer; the methyl carbon of acetate is incorporated into AMC. As can be seen in Fig. 1, the specific activity of the AMC obtained from methyl-labeled acetate should be greater than the specific activity of the pyruvate; this was not the case. However, while there is evidence that AMC is f&mea from substrate via pyruvate when ac-3 sp is incubated with’glucose (Table I), there is no evidence for a simple precursorproduct relatianship between pyruvic acid and AMC on incubation of UC-Ssp mycelium with acetate, since in this caseAMC appears and then disappears while pyruvic acid is still accumulating. The appearance and subsequent disappearance of AMC on incubation with acetate indicates that some other unknown process occurs, although acetate undoubtedly contributes partially to AMC formation via pyruvate. However, it seems qualitatively significant that only the methyl carbon of acetate enters the AMC, and these results are therefore evidence that acetate is metabolized by a cyclic mechanism. The greater incorporation of NaHCY03 into pyruvic acid than into AMC when UC-S.spmycelium is shaken with glucose and labeled bicarbonate is also consistent with the idea that pyruvate is formed from acetate via a cyclic mechanism. The incorporation of CY402into pyruvic acid can be explained as due to CO2 fixation into oxalacetic acid by combination with pyruvate and randomization of the label by conversion of oxalacetic acid to succinate with a subsequent return to pyruvate. These well-established reaction mechanisms would led to C1402incorporation into the carboxyl of pyruvate and would therefore not lead to incorporation of Ci4 into AMC. If acetate metabolism does occur by a cyclic mechanism in resting cultures, then it is more likely that the cycle is a,tricarboxylic rather than a clicarboxylic acid cycle. Sodium fluoroacetate acts in other organisms by conversion to fluorocitric acid which is an inhibitor of citric acid metabolism (17). The fluoroacetate inhibition of acetate and glucose oxidation by resting cultures of Neurospora and the accumulation of citric acid in the presence of fluoroacetate in both growing and resting cultures are therefore evidence for the utilization of a tricarboxylic acid cycle in Neurospora. Furthermore, the amount of pyruvic acid accumulated by the acetate-requiring mutant when oxidizing acetate is only about 1 y0 of the amount of acetate taken up on a molar basis. Since the ac sp strain used has a nearly complete block in its ability to oxidize pyruvic acid (6), nearly stoichiometric amounts of pyruvic acid would be expected to accumulate if pyruvate were an intermediate in acetate oxidation as required by the dicarboxylic acid cycle. Finally, if pyruvic
METSBOLISM
OF
ACETATE
91
acid were a necessary intermediate in acetate oxidation, then the CK-3sp strain used should be unable to eliminate the methyl of acetate as COZ as readily as the wild-type which has no difficulty in metabolizing pyruvate. Both wild-type and UC-S sp strains do eliminate the methyl of acetate as COZ at equivalent rates and both take up about t’he same amount of oxygen as a result of acetate oxidation at about the same rate. These data therefore eliminate pyruvate as an inbermediate in any mechanism of acetate oxidation by Neurospora. The tricarboxylic acid cycle best fulfils the requirements for a cyclic mechanism of acetate oxidation (to account for the incorporation of t,he methyl carbon of acetate into pyruvate more readily t’han the carboxyl carbon and to account for the failure of the carboxy carbon of acetate to be incorporated into AMC although the methyl carbon is incorporated) which includes citrate as an intermediate (fluoroacetate inhibition) but which does not include pyruvic acid as an intermediat’e. Pyruvate accumulation as a result of acetate oxidation would then be due to “leakage” from the cycle as a result of the breakdown of oxalacetic acid or malic acid. It is not possible to delineate the lesion in the single-gene “succinate” mutants. Failure of a strain of Neurospora to respond to cit,rate (5) is not) necessarily significant since the wild-type itself cannot utilize this compound as a carbon source. It is therefore not possible to decide at present whether the lesion in t)he “succinate” mutants occurs at the level of citrate formation or at the level of the conversion of citrate to Lu-ket)oglutaric acid. Even though these strains do respond adaptively to small quantities of acetate, it is obvious that they are able to make acetate at some stage of their life cycle; strains which do have real difficulty in synthesizing acetate require 10-20 t’imes the quantity of acetate required to give maximal growth of the ‘%uccinat,e” mutants, and the one UCmutant, t’hat responds t’o succinate requires relatively large amounts of this compound. If t)he evidence presented above for the occurrence and use of the tricarboxylic acid cycle as the major route for acetate oxidation by resting cultures of Neurospora is correct, then the “succinate” mutant,s should be unable to use acetate as a growth supplement unless some additional mechanism operates. Either the tricarboxylic acid cycle is blocked only during part of the growth cycle, or some supplementary mechanism for acetate oxidation such as the dicarboxylic acid cycle becomes available upon the addition of acetate or a dicarboxylic acid. It is
92
BERNARD
S. STRAUSS
not possible to decide between these alternatives at present, but either one makes unnecessary the assumption, seemingly required from the growth data, that the “succinate” mutant.s are unable to synthesize acetate. Inhibition of Pyruvic Acid Accumulation Several factors are involved in the interpretation of pyruvic acid accumulation by the acetate-requiring mutants; the sodium-ion content of the medium &ects pyruvate formation from the endogenous reserves of the mycelium (Table VIII), pyruvate is accumulated as a result of the metabolism of glucose or acetate (Table I), CO2 is incorporated into pyruvate (Table VI), and the presence of acet,ate reduces pyruvic acid accumulation from glucose (Table II). The reduction of pyruvic acid accumulation from glucose in the presence of acetate is a true inhibition of pyruvic acid formation by acetate or a closely related substance since (a) inhibitors of acetate metabolism do not restore the accumulation of pyruvate as they should if a metabolic product of acetate were responsible for the effect or if the fact of acetate metabolism alone were enough to depress the accumulation of pyruvate; (b) succinate, which is metabolized via pathways similar to that of acctate, does not reduce pyruvate accumulation; and (c) in the presence of acetate the specific activity of the respiratory CO2 produced from glucase-l-Cl4 is reduced to a greater extent in the ae sp mutant than in the wild-type (Table V). At the very least, this does not support the idea that acetate in some way promotes the metabolism of pyruvate. It is also difficult to understand how the inhibitory effect of fluoroacetate on pyruvic acid accumulation (Table III) is related to its inhibition of acetate metabolism, and this may therefore be a case in which fluoroacetate does not act by prior conversion to fluorocitrate. The phenomenon of the inhibition of precursor accumulation by the substance required for growth has been discussed by Adelberg and Umbarger (18) and by Cohn and Monod (19). Although the mechanisms differ in the various casesanalyzed, this inhibition by feedback seemsto be a method by which the organism can regulate the direction of its metabolic pathways. It does not seem that acetate inhibition of pyruvic acid formation is an inhibition of protein synthesis as suggested by Cohn and Monod (19) for tryptophan and methionine biosynthesis since inhibition is obtained in “resting” cultures.
METABOLISM OF ACETATE
93
ACKNOWLEDGMENTS The aut,hor would like to acknowledge the technical assistance of Mr. George Shaheen, hlr. Joseph Zizzi, and Miss M. L. Jezyk in the course of these investigations.
SUMMARY
1. Resting cultures of an acetate-requiring mutant accumulate pyruvie acid and acetylmethylcarbinol when shaken in buffer with glucose or acet,ate. 2. The methyl carbon of acetate is incorporated into the pyruvate and the acetylmethylcarbinol; the carboxyl carbon enters the pyruvate but not the acetylmethylcarbinol. Neurospora can incorporate COt into pyruvic acid. 3. Sodium fluoroacetate inhibits acetate oxidation and mycelial growth. Fluoroacetate-inhibited cultures accumulate citrate. 4. The met.hyl (barbon of acetate appears as CO2 as readily in acetaterequiring mutants which are unable to oxidize pyruvic acid as in the wild-type. 3. “Succinate” mut’ants Iv-ill grow adaptively on much smaller quantities of acetate t,han required by the ‘(acetate” mutants. 6. It is considered likely that Neurospora ordinarily oxidizes acetate via a t’ricarboxylic acid cycle, but that a dicarboxylic acid cycle might operat’e under certain conditions. 7. Acetate inhibits the accumulation of pyruvate from glucose by acetate-requiring mut)ant.s. REFERENCES 1. KREBS, H. A., GURIN, S., .4x11 EGC~LESTOX, I,. V., Hiochot~. J. 61, 614 (1!152). 2. ROBERTS, R. B., COIVIE, D. B., BRITTEN, R., BELTOX, E., .~NI) ABELSOS, I’. II., Proc. Natl. Acad. Sci. U. S. 39, 1013 (1953). 3. FOSTER, J. W., CARSON, S. F., ANTHONY, D. S., DAVIES, J. B., JEFFERSON, w. E:., ANU Loiw, i% v., Proc. 2C’atl. Acad. sci. I’. 8. 36, 1013 (1949). 4. BARROX, E. S. G., AND GHIRETTI, F., Biochim. et Biophys. Actn 12, 239 (1953). 5. LEWIS, R. W., Am. J. Botany 36, 292 (1948). 6. STRACSS, B. S., AND PIEROG, S., J. Gen. Microbial. 10. 221 (1954). 7. DUBES, GEORGE R., Ph.D. Thesis. California Institute of Technology, June, 1953. 8. BEADLE, G. W., AND TATC’M, E. I,., Am. J. Botany 32, 678 (1945). !I. FRIEDEMAKN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 10. WESTERFELD, W. W., J. Rio/. Chem. 161, 495 (1945).
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BERNARD
S. STRAUSS
11. GREEN, D. E., WESTERFELD, W. W., VENNESLAND, B., AND KNOX, W. E., J. Bid. Chem. 146, 69 (1942). 12. ETTINGER, R. H., GOLDBAUM, L. R., AND SMITH, L. H., JR., J. Biol. Chem. 199,
531 (1952). 13. JOHNSON, M. T., Science 94, 200 (1941). 14. STRAUSS, B. S., Arch. Biochem. and Biophys. 36, 33 (1952). 15. LEIN, J., APPLEBY, D., AND LEIN, P., Arch. Biochem. and Biophys. 34, 72 (1951). 16. SHEMIN, D., AND WITTENBERG, V., J. Biol. Chem. 192,315 (1951). 17. PETERS, R. A., Proc. Roy. Sot. (London) B139, 143 (1951). 18. ADELBERG, E. A., AND UMBARGER, H. E., J. Biol. Chem. 206, 475 (1953). 19. COHN, M., AND MONOD, J., Sot. Gen. Microbial. Symposia 3, 132 (1953).