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Biochemistry of filamentous fungi. III. Alternative routes for the breakdown of glucose by Fusarium lini
.\RCHI\‘F:S
OF
BIOCHETdISTRY
AND
BIOPIIYSICS
64, 80-87 (1956)
Biochemistry of Filamentous Fungi. III. Alternative Routes for the Breakdown of Glucose by Fusarium
linil
E. C. Heath,? DeLill Nasser, and Henry Koffler From the Laboratories Sciences, Purdue
of Bacteriology, Department University, West Lafayette,
Received
January
of Biological Indiana
26, 1956
INTRODUCTION
The observation (1) that growing cells of the strictly aerobic fungus Penicillium chrysogenum met,abolize at least tIT-o-thirds of the glucose utilized via an oxidative pathway3 (possibly the hexose monophosphate pathway) led to an extension of this work with t,he fermentativc mold Fusarium Zini. This fungus is int’eresting because it is one of a relatively few that, like some yeasts, arc capable of fermeniing certain hexoses to ethanol and CO2 in t#he ratio of 1: 1 (Z-4). Relatively little is known of the manner ill which this organism deals with glucose under aerobic conditions, although the data by Gould ct al. (4) \tith PusaGwz sp. II suggest t.hat different pathways may be operative depending upon t’hc presence of oxygen. These workers showed that the anaerobic production of CO2 by this organism is inhibited by low concentrations of fluoride and iodoacetate, nhilc t,he aerobic liberation of CC)2is relat,ircly insensitive to t,hcse inhibitors. The data presented in t,his paper have led to the conclusion that If’. 1 Supported in Ixtrt by the U. S. Atomic Energy Commission and the PAi I,ill> ;md Compsny. l’resentcd :>s :t portion of n pnpcr before t,he 111~1 Intermttiomd Congress of Biochemistry at Brussels, Belgium, on Augllst Znd, 19.5. 2 1’ostdoctor:Lte Fellow of the Lift Insurance SIedic:tl Research Fund. Present address: National Institute of Arthrit,is rind Met:lbolic I)ise:rscs, N:lt,ion:d Institutes of Healt~h, 13cthesdn, hlaryl,znd. 3 The term “osidative p:Lt,hwa)” in this p:lper is me:rnt to imply t,h:Lt t,he first carbon at.om of glucose is the first to lx liberated :IS CO? ; it is not llsetl with COW notations ns to the mech:&5m (or mcchnnisms) l)y which t,his oritlxtive decart,osylntion is accomplished. SO
ALTERXATIVE
ROUTES
XATERL~LS
OF GLKCOSE
Am
BKE.lRDOXYi
81
METHODS
The organism used in these studies was Fuswiu~ lini A.T.C.C. 9593. Soil stocks and inocula were prepared essentially as described for P. ch~!/soge?~zcmby Stout and Koffler (5). To ensure homogeneity, the spore suspension was shaken jvith -~-mm. diameter glass beads at a rate of 620 t,\vo and :I half-inch strokes/min. for 5 min. medium similar t,o that of GilJbs The organism was grown either in :L “complex” et al. (6), cscept that glucose was substit,uted for sylose, or in the “simple” mcdium used for 2’. chr~sogsnw~~ as described by Stout and Kofller (5). l'we~~ly-five milliliters of medium was added to each 125.ml. I’:rlenmeycr flask: the inoculated flasks were placed on :L reciprocating shaking machine (88 four-inch strokes/min.) at 26.-28°C. Cells were grown on t,he complex medium for 4S hr., then hnrvest,ed mcl washed according to the procedure of Gibbs el nl. (6). The duration of the growth period for cells cultured on the simple medium varied, and is indicated in the test. Oxygen uptake and CO* evolution were determined as described by Umbreit, Burris, and Staufrer (7). Respiratory C”0, was tr:tpped in Warburg or Erlenmcyer flasks, and the radioactivit,y was determined as described by Heath and Koffler (1). Radioactive samples were counted in a Tracerlab windowless gas-flex counter to an accuracy of f5y0, and corrected to zero self-absorption according to the method of Schweitzer and Stein (8). EXhanol was isolated from fermentation mixtures and converted to acetic acid according to the prorcdurc of Gibbs ELtrl. (6). ketic acid was degraded by the method of Phares (9). Glucose-I-CY4, -2.C14, and -6-C’” were kindly supplied by Dr. H. S. Isbell of the National Bureau of Standards. Uniformly labeled glucose (glucose-U-P) was purchased from bhc Nuclear Instruments and Chemical Company, Chicago, Illinois. Glucose-3,4-C’4 was prcpnrcd from rat liver glycogen as described by Nasser (10).
RESULTS Cells of F. hi were grown from a spore inoculum in flasks fitted with NaOH traps (1) in t’he simple medium in which either glucose-l-C’4, -2-C14, -3 ,4-C14, -6-P, or -U-Cl4 was the main source of carbon; glucose5-V was not available. The C1”OZwas collected for various periods during the 44 hr. of growth, and its radioactivit’y n-as det,ermined. As is shown in Fig. 1, the first carbon of glucose appears in the CO2 at a greater rate throughout the entire growth period t’han any of t,he other carbon atoms under scrutiny. This suggests that an oxidative pathn-ay funct.ions in this organism. To minimize the possibility of randomizat’ion which may be encoun-
82
HEATH,
NASSER
AND
KOFFLER
d4 RELEASED (CPM/Mg
EaCO&
HOURS 1. Oxidation of various carbon atoms of glucose to COa during the growt,h of Fztsarium Zini. Twenty-five milliliters of simple medium was used in each 125.ml. Erlenmeyer flask. Each flask contained 0.125 g. of specifically labeled glucose, representing 294,000 counts/min. (cpm) in the case of glucose-U-Cr4. A series of five flasks was set up for each specifically labeled sugar. The contents of the first mere analyzed for glucose and total radioactivity; the others n-ere inoculated with 0.5 ml. of a spore suspension of F. lini. Stoppers with attached CO2 traps containing 0.5 ml. of 40$& NaOH were inserted into the neck of the Aasks, and the flasks were incubated at 26°C. on a reciprocating shaking machine. At the end of 10 hr. of growth, 1 ml. of TO-‘i27c perchloric acid was injected through a diaphragm by means of a syringe and needle into one flask of each series; these flasks were shaken for at least another hour. At the same time, fresh CO, traps were placed into another flask of each series. The last two steps were repeated with other flasks at 20, 30, and 44 hr. The solution of iXaOH containing the Cl”O, was collected, and the t,rnpped C402 was precipitated and determined as BaC1+03 as described by Heath and Iiofhcr (1). Since the amount of CO, released was shown to be as accurate a measure of growth as the increase in mycelium, the weight of BaCOB was used as a criterion of growth in these experiment.s. The amount of radioactivity in the BaC103 was corrected on the basis of equivalent initial specific activity. The above experiment was repeated several times with essentially the same results. FIG.
ALTERNATIVE
ROUTES
OF
GLUCOSE
83
BREAKDOWN
tered during long periods of growth on a labeled substrate, and the resulting errors of interpretation, a similar experiment was performed except that briefer experimental periods were used. Cells were grown in the simple medium until they were in their most, active growth phase, and then were harvested (at 35 hr.) and washed as usual. The cells were resuspended in fresh, simple medium containing twice the concentration of nutrients but no glucose, and placed in Warburg flasks. Dilution by other componentjs of the react)ion mixture resulted in approximately normal concentrations of medium constituents. At the beginning of the experiment either glucose-l-P, -2-C14, -3,4-C’“, or -G-W was tipped into each flask from the side arm. After 1, 5, and 8 min., perchloric acid was added to duplicate flasks from t’hc other side arm to kill the cells. The C1402 released was collected and analyzed as indicated above. The results of t,his experiment are shown in Table I. The minimal participation of an oxidative pathway during the growth of t’he organism on the simple medium was evaluated by a comparison of the amount of radioact’ivity appearing in the CO, from glucose-l-C’* and from glucose-U-C’? (1). It was found that during the period of most rapid growth (30-44 hr.) a minimum of 17 % of the glucose utilized was channeled through such a pathway. The manner in \\-hich F. l&i metabolizes glucose under aerobic and anaerobic conditions was compared in the following way: Cells were harvestled after 48 hr. of growth in t,he complex medium, mashed, and TABLE Oxidation During
I
of Various Carbon Atoms of Glucose to CO2 Brief Periods of Growth of Fusarium lini
Experimental design was similar to that given in Fig. 1 in that specifically labeled glucose was used and the CO, formed was collected and counted as described. However, the reactions took place in Warburg flasks. XInin compartment: 1.5 ml. of a suspension of cells grown for 35 hr. on t.he simple medium (cells were resuspended in fresh double-strength simple medium minus glucose) and 0.6 ml. water; side arm: 0.5 ml. (5 pmoles; 102,750 counts/min.) of variously labeled glucose (added at 0 time); second side arm: 0.4 ml. of 70-72$& perchloric acid (added at 1, 5, or 8 min.) ; center well : 0.2 ml. of 40’% N&H. Gas phase : air. Radioactivity Glucose (Cl4 in position) 1
2 3,4 6
1
350 125 25 25
in COr , counls/min. Minutes 5
2,400 975 250 200
8
4,825 2,400 525 400
84
HEATH,
NASSER
AND
KOFFLER
resuspended in fresh medium minus glucose. The experimental design was essent,ially the same as ~#hnt,of the short-term expcrimcllt. rnclrtionctl before, differing only in that illstwd of the simple mctlilml untlcr aerobic conditions the c~~mplcs medium was ~mployccl 111ltl~rboth alr:lrrol)ic: :w well as aerobic conditions (lillal concclltrat ioll of the complcs medium was approximately half-strength). l?igurc 2 sho\\~s ihc reslllls of SU~ll ml experiment. In the presence of oxygen, the (‘-I of glucose is more readily oxidized to CO2 t,han carbon atoms 3 and 4, as was obacrved before. The reverse is t,rue when oxygen is absent. Similar results also are obtained when “resting” wlls, previously cultured on t’he complex medium for 48 hr., mdwbolizc specifically lab&d sugars aerobically and anucrobically. Table 11 summarizes t!hc dxt,a obtained from two such cxperimellts. Under aerobic conditions it is t,hc first carbon atom t’hat is most rapidly oxidized t,o CO, Under anaerobic conditions the (102 arises preferent.ially from (wbon atoms 3 and 4, as AEROBIC
ANAEROBIC
CPM IN
co2
0
5
IO
0
5
IO
MINUTES
FIG. 2. Oxidation of glucose-l-Cl4 and -3,4-C” to CO, by Fusariwn lini under aerobic and anaerobic conditions. Main compartment: 2.1 ml. (0.1 g. wet weight) of a suspension in fresh complex medium minus glucose of cells grown for 48 hr. on the complex medium; side arm: 0.5 ml. [5 pmoles; 30,775 countsjmin. (cpm)] of either glucose-l-Cl4 or glucose-3,4-C’” (added at 0 time); second side arm: 0.4 ml. of 70-720/, perchloric acid (added after 5 or 10 min.); center well: 0.2 ml. of 40% NaOH. Gas phase: aerobic, air; anaerobic, 99.6% h’, (by flushing for 25 min.).
ALTERNATlVE
ROUTES
OF
GLUCOSE
TABLE
8' 3
BREAKDOWK
II
of Variously Labeled Sugars to CO, by “Resting” Cells of Oxidation Fusarium lini under Aerobic and Anaerobic Conditions
Main compartment: 2.1 ml. (0.1 g. wet weight) of a suspension in M/15 phosphate buffer, pII 6.4, of cells grown for -IS hr. on the comgles medium; side arm: 0.5 ml. (5 pmoles; 57,000 counts/nun.) of variously labeled glucose (added at 0 time); second side arm: 0.4 ml. of 70-72y0 perchloric acid; cent,er well: 0.2 ml. of 40% Sa0I-I. Gas phase: aerobic, air; anaerobic, 99.69?c N, (by flushing). Experiment # 1 was allowed to proceed for 20 min., ICxpt. #2 for 40 min. Radioactivity
in COz , counlslmin.
81
#2
Glucose (Cl4 in position)
-02
$02
1-6 1 2 3,4 G
550 125 25 650 75
3,400 5,300 1,775 2,650 550
-02
2,625 1,075 250 2, G75 125
+a
S, 825 20 ) 750 7,150 4,825
5,200
it ~vould if the organism fermented glucose via the reactions of the EMP scheme; however, the concurrent participation of a pathway involving t’he preferential liberation of C-l is also indicated. The ethanol formed from glucose-l-CYA was isolated in another experiment, which was allowed to proceed until all of t,he 5 ~moles of glucose added init’ially had been utilized. An aliquot was degraded in a steplvise manner to determine the position of the radioactivity in the molecule. 1111of the radioactivity contained in t,he aliquot degraded (660 counts/min.) occurred in the methyl position of the compound. The ethanol formed from glucose3,4-C?” was unlabeled. ‘These data support the premise that the et,hanol is formed via the EMP mechanism.
The experiments described in this paper do not deal with the enzymatic details concerning the breakdown of glucose by F. lini. However, the data obtained indicate that under anaerobic conditions this organism degrades glucose mainly, but perhaps not entirely, in the manner depictcd by the EhlP scheme. Conclusions rcgardirlg t,he existence of a fermentat,ive pat,hrvay in FzlsarizinL not involving phosphorylated intermediate compounds (11) riced to be reconsidered in t,he light of the information presented in this paper and our experience that various phosphorylat.ed compounds (glucose-l-phosphate, glucose-6-phosphate, fructose-l, 6-diphosphate, ribose-5-phosphat,e, adenosinc monophosphate,
86
HEATH,
NASSER
AND KOFFLER
and adenosine diphosphate) can be isolated from the mycelium of this organism.4 Under aerobic conditions F. Zini apparently is capable of utilizing an oxidative pathway, perhaps the hexose monophosphate pathway, by which the C-l of glucose is the first carbon atom to be released as COZ . In all experiments in which air was used as the gaseous phase, C-l was liberated as CO, more rapidly than any of t,he other carbon atoms under observation. The finding of Gould cl al. (4) that the aerobic met,abolism of glucose by Fusarium sp. H is inhibited by fluoride and iodoncetate to a much lesser degree than is the fermentation of glucose is consistent with the assertion that this organism relies more heavily on one pathway or another, depending upon the presence of oxygen. Apparently, the effect of oxygen in influencing t,he extent to which alternative pathways are being used in Fusarium is similar to t,hnt shown for the yeast Xaccharomyces cerevisiae, lvhich fermems glucose via t,he EMP pathway but also uses an oxidative pathway in its aerobic metabolism (12, 13). It must be remembered that the real participation of an oxidative pathway during growth may be considerably greater than t,he estimate regarding the minimal value (ea. 20 %). This seems likely because CO2 is released strikingly more rapidly from the first carbon of glucose than from any of the other carbon atoms. IIeat,h and Koffler (1) discussed the limitations of such estimat.es in a paper t*hat presents the details for such calculations and also a more complete explanation of data that are similar in nature to those given here. SUMMARY
Fusarium lini in the absence of oxygen is capable of ferment’ing glucose probably as represented by the Embden-Meyerhof-Parnas scheme; in the presence of oxygen this organism apparent’ly utilizes an oxidative pathway. These assertions are supported by the following observations: Under anaerobic conditions “rcst’ing” cells metabolizing glucose release COZ most readily from the 3rd and 4th carbon atoms; the ethanol formed from glucose-l-C4 is methyl-labeled, while that formed from glucose3 ,4-Cl4 is unlabeled. Under aerobic conditions both growing and “resting” cells oxidize the first carbon atom of glucose to CO2 more readily than any of the other atoms. 4 The latter data will he published in full upon completion of the study, which is still in progress. Also the data of Cochrane [Cochranc, V. W., Mycologia 48, 1 (1956)], which appeared after this paper had been submitted for publication, support the view that F. Zini ferments glucose in accordance with the EMI’ pathway.
Si
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. '3. 10. Il. 12.
HEATH, E. C., AND KOFFLER, II., J. Bacterial. 71, 174 (1856). NORD, Ii’. F., Ergeb. Enzpforsch. 8, 149 (1030). (10%). NORD, F. F., Daarx\sx, E., AND HOFSTETTER, H., Biochcnz. Z. 285,241 GOULD, 13. S., AND TYTELL, A. il., .I. Gen. Physiol. 24, 655 (1941). STOUT, H. A., APEDIIULL, 11. I'., Lldmncc.s i?a E'nz!pml. 5, 165 (1945). I~LliMICSTII.\I,, 1%..J.> LK\~~s, IC. F.. \ST> V'IGINIIOT.SE, S.; .I. .-!n?. f”hff?~. SOC. 76, 6093 (1054).
OF
BIOCHETdISTRY
AND
BIOPIIYSICS
64, 80-87 (1956)
Biochemistry of Filamentous Fungi. III. Alternative Routes for the Breakdown of Glucose by Fusarium
linil
E. C. Heath,? DeLill Nasser, and Henry Koffler From the Laboratories Sciences, Purdue
of Bacteriology, Department University, West Lafayette,
Received
January
of Biological Indiana
26, 1956
INTRODUCTION
The observation (1) that growing cells of the strictly aerobic fungus Penicillium chrysogenum met,abolize at least tIT-o-thirds of the glucose utilized via an oxidative pathway3 (possibly the hexose monophosphate pathway) led to an extension of this work with t,he fermentativc mold Fusarium Zini. This fungus is int’eresting because it is one of a relatively few that, like some yeasts, arc capable of fermeniing certain hexoses to ethanol and CO2 in t#he ratio of 1: 1 (Z-4). Relatively little is known of the manner ill which this organism deals with glucose under aerobic conditions, although the data by Gould ct al. (4) \tith PusaGwz sp. II suggest t.hat different pathways may be operative depending upon t’hc presence of oxygen. These workers showed that the anaerobic production of CO2 by this organism is inhibited by low concentrations of fluoride and iodoacetate, nhilc t,he aerobic liberation of CC)2is relat,ircly insensitive to t,hcse inhibitors. The data presented in t,his paper have led to the conclusion that If’. 1 Supported in Ixtrt by the U. S. Atomic Energy Commission and the PAi I,ill> ;md Compsny. l’resentcd :>s :t portion of n pnpcr before t,he 111~1 Intermttiomd Congress of Biochemistry at Brussels, Belgium, on Augllst Znd, 19.5. 2 1’ostdoctor:Lte Fellow of the Lift Insurance SIedic:tl Research Fund. Present address: National Institute of Arthrit,is rind Met:lbolic I)ise:rscs, N:lt,ion:d Institutes of Healt~h, 13cthesdn, hlaryl,znd. 3 The term “osidative p:Lt,hwa)” in this p:lper is me:rnt to imply t,h:Lt t,he first carbon at.om of glucose is the first to lx liberated :IS CO? ; it is not llsetl with COW notations ns to the mech:&5m (or mcchnnisms) l)y which t,his oritlxtive decart,osylntion is accomplished. SO
ALTERXATIVE
ROUTES
XATERL~LS
OF GLKCOSE
Am
BKE.lRDOXYi
81
METHODS
The organism used in these studies was Fuswiu~ lini A.T.C.C. 9593. Soil stocks and inocula were prepared essentially as described for P. ch~!/soge?~zcmby Stout and Koffler (5). To ensure homogeneity, the spore suspension was shaken jvith -~-mm. diameter glass beads at a rate of 620 t,\vo and :I half-inch strokes/min. for 5 min. medium similar t,o that of GilJbs The organism was grown either in :L “complex” et al. (6), cscept that glucose was substit,uted for sylose, or in the “simple” mcdium used for 2’. chr~sogsnw~~ as described by Stout and Kofller (5). l'we~~ly-five milliliters of medium was added to each 125.ml. I’:rlenmeycr flask: the inoculated flasks were placed on :L reciprocating shaking machine (88 four-inch strokes/min.) at 26.-28°C. Cells were grown on t,he complex medium for 4S hr., then hnrvest,ed mcl washed according to the procedure of Gibbs el nl. (6). The duration of the growth period for cells cultured on the simple medium varied, and is indicated in the test. Oxygen uptake and CO* evolution were determined as described by Umbreit, Burris, and Staufrer (7). Respiratory C”0, was tr:tpped in Warburg or Erlenmcyer flasks, and the radioactivit,y was determined as described by Heath and Koffler (1). Radioactive samples were counted in a Tracerlab windowless gas-flex counter to an accuracy of f5y0, and corrected to zero self-absorption according to the method of Schweitzer and Stein (8). EXhanol was isolated from fermentation mixtures and converted to acetic acid according to the prorcdurc of Gibbs ELtrl. (6). ketic acid was degraded by the method of Phares (9). Glucose-I-CY4, -2.C14, and -6-C’” were kindly supplied by Dr. H. S. Isbell of the National Bureau of Standards. Uniformly labeled glucose (glucose-U-P) was purchased from bhc Nuclear Instruments and Chemical Company, Chicago, Illinois. Glucose-3,4-C’4 was prcpnrcd from rat liver glycogen as described by Nasser (10).
RESULTS Cells of F. hi were grown from a spore inoculum in flasks fitted with NaOH traps (1) in t’he simple medium in which either glucose-l-C’4, -2-C14, -3 ,4-C14, -6-P, or -U-Cl4 was the main source of carbon; glucose5-V was not available. The C1”OZwas collected for various periods during the 44 hr. of growth, and its radioactivit’y n-as det,ermined. As is shown in Fig. 1, the first carbon of glucose appears in the CO2 at a greater rate throughout the entire growth period t’han any of t,he other carbon atoms under scrutiny. This suggests that an oxidative pathn-ay funct.ions in this organism. To minimize the possibility of randomizat’ion which may be encoun-
82
HEATH,
NASSER
AND
KOFFLER
d4 RELEASED (CPM/Mg
EaCO&
HOURS 1. Oxidation of various carbon atoms of glucose to COa during the growt,h of Fztsarium Zini. Twenty-five milliliters of simple medium was used in each 125.ml. Erlenmeyer flask. Each flask contained 0.125 g. of specifically labeled glucose, representing 294,000 counts/min. (cpm) in the case of glucose-U-Cr4. A series of five flasks was set up for each specifically labeled sugar. The contents of the first mere analyzed for glucose and total radioactivity; the others n-ere inoculated with 0.5 ml. of a spore suspension of F. lini. Stoppers with attached CO2 traps containing 0.5 ml. of 40$& NaOH were inserted into the neck of the Aasks, and the flasks were incubated at 26°C. on a reciprocating shaking machine. At the end of 10 hr. of growth, 1 ml. of TO-‘i27c perchloric acid was injected through a diaphragm by means of a syringe and needle into one flask of each series; these flasks were shaken for at least another hour. At the same time, fresh CO, traps were placed into another flask of each series. The last two steps were repeated with other flasks at 20, 30, and 44 hr. The solution of iXaOH containing the Cl”O, was collected, and the t,rnpped C402 was precipitated and determined as BaC1+03 as described by Heath and Iiofhcr (1). Since the amount of CO, released was shown to be as accurate a measure of growth as the increase in mycelium, the weight of BaCOB was used as a criterion of growth in these experiment.s. The amount of radioactivity in the BaC103 was corrected on the basis of equivalent initial specific activity. The above experiment was repeated several times with essentially the same results. FIG.
ALTERNATIVE
ROUTES
OF
GLUCOSE
83
BREAKDOWN
tered during long periods of growth on a labeled substrate, and the resulting errors of interpretation, a similar experiment was performed except that briefer experimental periods were used. Cells were grown in the simple medium until they were in their most, active growth phase, and then were harvested (at 35 hr.) and washed as usual. The cells were resuspended in fresh, simple medium containing twice the concentration of nutrients but no glucose, and placed in Warburg flasks. Dilution by other componentjs of the react)ion mixture resulted in approximately normal concentrations of medium constituents. At the beginning of the experiment either glucose-l-P, -2-C14, -3,4-C’“, or -G-W was tipped into each flask from the side arm. After 1, 5, and 8 min., perchloric acid was added to duplicate flasks from t’hc other side arm to kill the cells. The C1402 released was collected and analyzed as indicated above. The results of t,his experiment are shown in Table I. The minimal participation of an oxidative pathway during the growth of t’he organism on the simple medium was evaluated by a comparison of the amount of radioact’ivity appearing in the CO, from glucose-l-C’* and from glucose-U-C’? (1). It was found that during the period of most rapid growth (30-44 hr.) a minimum of 17 % of the glucose utilized was channeled through such a pathway. The manner in \\-hich F. l&i metabolizes glucose under aerobic and anaerobic conditions was compared in the following way: Cells were harvestled after 48 hr. of growth in t,he complex medium, mashed, and TABLE Oxidation During
I
of Various Carbon Atoms of Glucose to CO2 Brief Periods of Growth of Fusarium lini
Experimental design was similar to that given in Fig. 1 in that specifically labeled glucose was used and the CO, formed was collected and counted as described. However, the reactions took place in Warburg flasks. XInin compartment: 1.5 ml. of a suspension of cells grown for 35 hr. on t.he simple medium (cells were resuspended in fresh double-strength simple medium minus glucose) and 0.6 ml. water; side arm: 0.5 ml. (5 pmoles; 102,750 counts/min.) of variously labeled glucose (added at 0 time); second side arm: 0.4 ml. of 70-72$& perchloric acid (added at 1, 5, or 8 min.) ; center well : 0.2 ml. of 40’% N&H. Gas phase : air. Radioactivity Glucose (Cl4 in position) 1
2 3,4 6
1
350 125 25 25
in COr , counls/min. Minutes 5
2,400 975 250 200
8
4,825 2,400 525 400
84
HEATH,
NASSER
AND
KOFFLER
resuspended in fresh medium minus glucose. The experimental design was essent,ially the same as ~#hnt,of the short-term expcrimcllt. rnclrtionctl before, differing only in that illstwd of the simple mctlilml untlcr aerobic conditions the c~~mplcs medium was ~mployccl 111ltl~rboth alr:lrrol)ic: :w well as aerobic conditions (lillal concclltrat ioll of the complcs medium was approximately half-strength). l?igurc 2 sho\\~s ihc reslllls of SU~ll ml experiment. In the presence of oxygen, the (‘-I of glucose is more readily oxidized to CO2 t,han carbon atoms 3 and 4, as was obacrved before. The reverse is t,rue when oxygen is absent. Similar results also are obtained when “resting” wlls, previously cultured on t’he complex medium for 48 hr., mdwbolizc specifically lab&d sugars aerobically and anucrobically. Table 11 summarizes t!hc dxt,a obtained from two such cxperimellts. Under aerobic conditions it is t,hc first carbon atom t’hat is most rapidly oxidized t,o CO, Under anaerobic conditions the (102 arises preferent.ially from (wbon atoms 3 and 4, as AEROBIC
ANAEROBIC
CPM IN
co2
0
5
IO
0
5
IO
MINUTES
FIG. 2. Oxidation of glucose-l-Cl4 and -3,4-C” to CO, by Fusariwn lini under aerobic and anaerobic conditions. Main compartment: 2.1 ml. (0.1 g. wet weight) of a suspension in fresh complex medium minus glucose of cells grown for 48 hr. on the complex medium; side arm: 0.5 ml. [5 pmoles; 30,775 countsjmin. (cpm)] of either glucose-l-Cl4 or glucose-3,4-C’” (added at 0 time); second side arm: 0.4 ml. of 70-720/, perchloric acid (added after 5 or 10 min.); center well: 0.2 ml. of 40% NaOH. Gas phase: aerobic, air; anaerobic, 99.6% h’, (by flushing for 25 min.).
ALTERNATlVE
ROUTES
OF
GLUCOSE
TABLE
8' 3
BREAKDOWK
II
of Variously Labeled Sugars to CO, by “Resting” Cells of Oxidation Fusarium lini under Aerobic and Anaerobic Conditions
Main compartment: 2.1 ml. (0.1 g. wet weight) of a suspension in M/15 phosphate buffer, pII 6.4, of cells grown for -IS hr. on the comgles medium; side arm: 0.5 ml. (5 pmoles; 57,000 counts/nun.) of variously labeled glucose (added at 0 time); second side arm: 0.4 ml. of 70-72y0 perchloric acid; cent,er well: 0.2 ml. of 40% Sa0I-I. Gas phase: aerobic, air; anaerobic, 99.69?c N, (by flushing). Experiment # 1 was allowed to proceed for 20 min., ICxpt. #2 for 40 min. Radioactivity
in COz , counlslmin.
81
#2
Glucose (Cl4 in position)
-02
$02
1-6 1 2 3,4 G
550 125 25 650 75
3,400 5,300 1,775 2,650 550
-02
2,625 1,075 250 2, G75 125
+a
S, 825 20 ) 750 7,150 4,825
5,200
it ~vould if the organism fermented glucose via the reactions of the EMP scheme; however, the concurrent participation of a pathway involving t’he preferential liberation of C-l is also indicated. The ethanol formed from glucose-l-CYA was isolated in another experiment, which was allowed to proceed until all of t,he 5 ~moles of glucose added init’ially had been utilized. An aliquot was degraded in a steplvise manner to determine the position of the radioactivity in the molecule. 1111of the radioactivity contained in t,he aliquot degraded (660 counts/min.) occurred in the methyl position of the compound. The ethanol formed from glucose3,4-C?” was unlabeled. ‘These data support the premise that the et,hanol is formed via the EMP mechanism.
The experiments described in this paper do not deal with the enzymatic details concerning the breakdown of glucose by F. lini. However, the data obtained indicate that under anaerobic conditions this organism degrades glucose mainly, but perhaps not entirely, in the manner depictcd by the EhlP scheme. Conclusions rcgardirlg t,he existence of a fermentat,ive pat,hrvay in FzlsarizinL not involving phosphorylated intermediate compounds (11) riced to be reconsidered in t,he light of the information presented in this paper and our experience that various phosphorylat.ed compounds (glucose-l-phosphate, glucose-6-phosphate, fructose-l, 6-diphosphate, ribose-5-phosphat,e, adenosinc monophosphate,
86
HEATH,
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AND KOFFLER
and adenosine diphosphate) can be isolated from the mycelium of this organism.4 Under aerobic conditions F. Zini apparently is capable of utilizing an oxidative pathway, perhaps the hexose monophosphate pathway, by which the C-l of glucose is the first carbon atom to be released as COZ . In all experiments in which air was used as the gaseous phase, C-l was liberated as CO, more rapidly than any of t,he other carbon atoms under observation. The finding of Gould cl al. (4) that the aerobic met,abolism of glucose by Fusarium sp. H is inhibited by fluoride and iodoncetate to a much lesser degree than is the fermentation of glucose is consistent with the assertion that this organism relies more heavily on one pathway or another, depending upon the presence of oxygen. Apparently, the effect of oxygen in influencing t,he extent to which alternative pathways are being used in Fusarium is similar to t,hnt shown for the yeast Xaccharomyces cerevisiae, lvhich fermems glucose via t,he EMP pathway but also uses an oxidative pathway in its aerobic metabolism (12, 13). It must be remembered that the real participation of an oxidative pathway during growth may be considerably greater than t,he estimate regarding the minimal value (ea. 20 %). This seems likely because CO2 is released strikingly more rapidly from the first carbon of glucose than from any of the other carbon atoms. IIeat,h and Koffler (1) discussed the limitations of such estimat.es in a paper t*hat presents the details for such calculations and also a more complete explanation of data that are similar in nature to those given here. SUMMARY
Fusarium lini in the absence of oxygen is capable of ferment’ing glucose probably as represented by the Embden-Meyerhof-Parnas scheme; in the presence of oxygen this organism apparent’ly utilizes an oxidative pathway. These assertions are supported by the following observations: Under anaerobic conditions “rcst’ing” cells metabolizing glucose release COZ most readily from the 3rd and 4th carbon atoms; the ethanol formed from glucose-l-C4 is methyl-labeled, while that formed from glucose3 ,4-Cl4 is unlabeled. Under aerobic conditions both growing and “resting” cells oxidize the first carbon atom of glucose to CO2 more readily than any of the other atoms. 4 The latter data will he published in full upon completion of the study, which is still in progress. Also the data of Cochrane [Cochranc, V. W., Mycologia 48, 1 (1956)], which appeared after this paper had been submitted for publication, support the view that F. Zini ferments glucose in accordance with the EMI’ pathway.
Si
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. '3. 10. Il. 12.
HEATH, E. C., AND KOFFLER, II., J. Bacterial. 71, 174 (1856). NORD, Ii’. F., Ergeb. Enzpforsch. 8, 149 (1030). (10%). NORD, F. F., Daarx\sx, E., AND HOFSTETTER, H., Biochcnz. Z. 285,241 GOULD, 13. S., AND TYTELL, A. il., .I. Gen. Physiol. 24, 655 (1941). STOUT, H. A., APEDI