THE METABOLISM OF SPECIES OF STREPTOMYCES VI. TRICARBOxyLIC ACID CYCLE REACTIONS IN STREPTOMYCE8 COELICOLOR VINCENT W. COCHRANE AND HARRY D. PECK, JR. Wesleyan University, Middletown, Connecticut Received for publication May 19,1952 In the preceding paper of this series (Cochrane, 1952) it was shown that bulk formation of sue- cinic acid by Streptomyces coelicolor (Muller) Waksman and Henrici is effected via reductive carboxylation of pyruvate or a derivative of it. There was no evidence that bulk formation of succinate results from a block in an oxidative pathway such as the tricarboxylic acid cycle. Nevertheless, it was pointed out that the evi- dence, being restricted to the problem of the origin of succinate, in no way bore on the opera- tion of such a cycle in the orgaimsm. Ajl (1951) has reviewed recently the problem of the existence of the tricarboxylic cycle in bac- teria and other microorganism, concluding with regard to bacteria that the cycle has been shown conclusively in only a few forms, and con- cluding further that there are some bacteria in which the full set of reactions does not occur. The same review points out that in the aerobic filamentous fungi some of the distinctive reactions of the cycle, e.g., the oxidative decarboxylation of a-ketoglutaric acid, have never been demon- strated. In the present work, the over-all respiratory pattern of S. coelicolor has been studied with whole cells and extracts. Since simple mano- metric data alone can never afford critical evi- dence, some of the key reactions have been in- vestigated chemically, and the incorporation of acetate into a-ketoglutarate has been followed with the aid of carbon-14. MATE}IA AND METHODS Streptomyces coelicolor was grown for these experiments in Difco nutrient broth supple- mented with glucose (0.04 m), potassium phos- phate buffer (0.033 m, pH 7.4), MgSO4 (0.001 M), and minor elements (Cochrane and Dimmick, 1949). Cultures were grown at 25 C on a recipro- cating shaker (stroke 9 cm, frequency 95 strokes per minute), except as noted otherwise. For manometric work with whole cells the pellets of growth were washed 3 times on a coarse sintered gla funnel with salt solution (K1HPO4, 0.003 M; MgSO4, 0.001 M), blended 30 seconds in 10 weights of water, and added by pipette to the respiration vessels. Cell-free preparations were made by grinding with 3.3 weights of powdered pyrex glass (40 mesh) in the alkaline isotonic KCl mixture of Potter (1948). Grinding was continued in a chilled mortar until a change in consistency indicated substantially complete destruction- usually about 10 minutes. Glass and cell debris were centrifuged out at 2,500 rpm in a Sorvall model XL centrifuge, and the supernatant, ad- justed to contain approximately 1 mg nitrogen per ml, was used in the Warburg or other vessel. Methods for use and determination of carbon- 14 were as previously described (Cochrane, 1952). Citrate was determined by the method of Weil- Malherbe and Bone (1949), pyruvate and a- ketoglutarate by the method of Friedemann and Haugen (1943), and acetate by the chromato- graphic method of Bueding and Yale (1951), as modified by Kohlmiller and Gest (1951). Con- ventional manometric methods were used with a bath temperature of 30 C. In experiments with whole cells and acidic substrates, a pH of 5.6 was maintained to facilitate penetration of substrate into the cell. Adenosine triphosphate, diphos- phopyridine nucleotide (65 per cent purity), thiamin pyrophosphate, and a-ketoglutaric acid were obtained from commercial sources. RESULTS Oxidation of carbon compounds by intact ceUs. In table 1 are collected data from several experi- ments on the respiratory capacity of whole celLs. The values are reported after subtraction of endogenous respiration. It is evident first that older cells are more ac- tive than younger, the effect of age (days from inoculation to harvest) being especially marked 137 on February 28, 2021 by guest http://jb.asm.org/ Downloaded from
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THE METABOLISM OF SPECIES OF STREPTOMYCES
VI. TRICARBOxyLIC ACID CYCLE REACTIONS IN STREPTOMYCE8 COELICOLOR
VINCENT W. COCHRANE AND HARRY D. PECK, JR.Wesleyan University, Middletown, Connecticut
Received for publication May 19,1952
In the preceding paper of this series (Cochrane,1952) it was shown that bulk formation of sue-cinic acid by Streptomyces coelicolor (Muller)Waksman and Henrici is effected via reductivecarboxylation of pyruvate or a derivative of it.There was no evidence that bulk formation ofsuccinate results from a block in an oxidativepathway such as the tricarboxylic acid cycle.Nevertheless, it was pointed out that the evi-dence, being restricted to the problem of theorigin of succinate, in no way bore on the opera-tion of such a cycle in the orgaimsm.
Ajl (1951) has reviewed recently the problemof the existence of the tricarboxylic cycle in bac-teria and other microorganism, concluding withregard to bacteria that the cycle has been shownconclusively in only a few forms, and con-cluding further that there are some bacteria inwhich the full set of reactions does not occur.The same review points out that in the aerobicfilamentous fungi some of the distinctive reactionsof the cycle, e.g., the oxidative decarboxylationof a-ketoglutaric acid, have never been demon-strated.
In the present work, the over-all respiratorypattern of S. coelicolor has been studied withwhole cells and extracts. Since simple mano-metric data alone can never afford critical evi-dence, some of the key reactions have been in-vestigated chemically, and the incorporation ofacetate into a-ketoglutarate has been followedwith the aid of carbon-14.
MATE}IA AND METHODS
Streptomyces coelicolor was grown for theseexperiments in Difco nutrient broth supple-mented with glucose (0.04 m), potassium phos-phate buffer (0.033 m, pH 7.4), MgSO4 (0.001 M),and minor elements (Cochrane and Dimmick,1949). Cultures were grown at 25 C on a recipro-cating shaker (stroke 9 cm, frequency 95 strokesper minute), except as noted otherwise.
For manometric work with whole cells thepellets of growth were washed 3 times on a coarsesintered gla funnel with salt solution (K1HPO4,0.003 M; MgSO4, 0.001 M), blended 30 seconds in10 weights of water, and added by pipette to therespiration vessels. Cell-free preparations weremade by grinding with 3.3 weights of powderedpyrex glass (40 mesh) in the alkaline isotonic KClmixture of Potter (1948). Grinding was continuedin a chilled mortar until a change in consistencyindicated substantially complete destruction-usually about 10 minutes. Glass and cell debriswere centrifuged out at 2,500 rpm in a Sorvallmodel XL centrifuge, and the supernatant, ad-justed to contain approximately 1 mg nitrogenper ml, was used in the Warburg or other vessel.
Methods for use and determination of carbon-14 were as previously described (Cochrane, 1952).Citrate was determined by the method of Weil-Malherbe and Bone (1949), pyruvate and a-ketoglutarate by the method of Friedemann andHaugen (1943), and acetate by the chromato-graphic method of Bueding and Yale (1951), asmodified by Kohlmiller and Gest (1951). Con-ventional manometric methods were used with abath temperature of 30 C. In experiments withwhole cells and acidic substrates, a pH of 5.6 wasmaintained to facilitate penetration of substrateinto the cell. Adenosine triphosphate, diphos-phopyridine nucleotide (65 per cent purity),thiamin pyrophosphate, and a-ketoglutaric acidwere obtained from commercial sources.
RESULTS
Oxidation of carbon compounds by intact ceUs.In table 1 are collected data from several experi-ments on the respiratory capacity of whole celLs.The values are reported after subtraction ofendogenous respiration.
It is evident first that older cells are more ac-tive than younger, the effect of age (days frominoculation to harvest) being especially marked
TABLE 1Respiration of intact cells of different ages
(Expressed as Qo2 (N)*)AGE OF czLL,t DAYS
SUBSTRATEt___- -
2 3 4
Glucose, 0.01 M 171 274 368Pyruvate, 0.01 m 42 86 108Acetate, 0.005 M -2 41 60Citrate, 0.01 M 14 0 5a-Ketoglutarate, 0.01 M 7 1 2Succinate, 0.01 m 62 78 103Fumarate, 0.01 m 10 12 74DL-Malate, 0.02 m 7 11 57Lactate, 0.01 m 14 38
(0.017 M phosphate, pH 5.6), substrate (adjustedto pH 5.6 with KOH), water to 3.2 ml. Gas phaseair, temperature 30 C.
t Days from inoculation to harvest; cells usedimmediately after harvest.
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with malate and acetate as substrates. Youngcells oxidize only glucose, pyruvate, and succinateof the compounds tested; it is significant that thesequence glucose-pyruvate-succinate occurs inthis organism and is established early in theculture cycle (Cochrane, 1952; Cochrane andDimmick, 1949). As cells become older, activityon these substrates increases, and other sub-strates (malate, lactate, and acetate) are metab-olized. In view of the importance of cell permea-bility (see below), we are inclined at present tobelieve that the age effect is in fact a permeabilityeffect although admittedly more critical experi-ments are needed to buttress this belief.
Representative respiratory data are plotted infigure 1, with the purpose of giving a picture of theactivity of intact cells. The high endogenousrate is characteristic of the actinomycetes andthe fungi (Woodruff and Foster, 1943; Stout andKoffler, 1951) and complicates interpretation ofmarginal rates of respiration. The cells used forthe experiment illustrated were 4 days old and at
0
TIME, MINUTES
Figure 1. Respiratory metabolism of intact cells of Streptomyces coelicolor. All flasks contained phos-phate buffer 0.017 M (pH 5.6), Mg+3.3 X 10-3 m, Mn 1.0 X 10- M, cell suspension 1 ml, water to 3.0ml. Substrates at 0.01 M, except DL-Malate (0.02 M) and acetate (0.005 m), tipped in 20 minutes beforestart of readings. Acid substrates neutralized with NaOH to pH 5.6. KOH in center well, gas phase air,temperature 30 C.
the peak of their respiratory activity. Even underthese favorable conditions, only with glucose isthe rate of respiration with substrate more thantwice endogenous. Attempts to reduce the endog-enous rate by starvation or prolonged aerationfailed in that reduction in the endogenous ratewas achieved only by such prolonged treatmentas to reduce proportionately or more than pro-
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in the filamentous fungi, such methods offer twoadvantages: elimination of permeability effectsand reduction of the endogenous respirationrate.
Oxidation of putative intermediates by cell-free preparations (figure 2) suggests immediatelya more consistent pattern of metabolism thancould be inferred from data on intact celLs. Both
TIME, MINUTES
Figure B. Respiratory metabolism of a cell-free extract of Streptomyces coelicolor. All flasks containedphosphate buffer 0.008 M (pH 7.3), Mg++ 3.3 X 10-3 M, Mn H 1.0 X 10-6 M, methylene blue 1.0 X 10-8 m,
extract 1.0 ml, water to 3.0 ml. Substrates at 0.01 M, except DL-malate (0.02 M), tipped in 10 minutesbefore start of readings. Supplements as shown: DPN (diphosphopyridine nucleotide) 5.9 X 10-3 M,ATP (adenosine triphosphate) 1.0 X 10-' M, TPP (cocarboxylase) 8.5 X 10-, m. KOH in center well, gas
phase air, temperature 30 C.
portionately the rate of oxidation of glucose andpyruvate.The data on intact cells, taken by itself, would
render very doubtful the applicability to thisorganism of any cyclic mechanism involvingcitrate or a-ketoglutarate, especially the latter.
Oxidation of carbon compounds by cell-free ex-
tracts. The disadvantages of intact cells as experi-mental material prompted a shift to cell-freemethods. In the actinomycetes and presumably
citrate and a-ketoglutarate are oxidized, theformer very rapidly. In the case of fumarate,data are included to show the stimulatory effectof diphosphopyridine nucleotide; the same stimu-lation has been observed with malate or succinateas substrate, suggesting that succinate andfumarate are metabolized at least in part viamalate. Rates of oxidation of the 4-carbon di-carboxylic acids are approximately equal. Thesefindings and the demonstration of enzymatic
decarboxylation of oxalacetate (table 2) are allconsistent with the operation of a tricarboxylicacid cycle.
In no case was it possible to obtain a cell-freepreparation capable of oxidizing pyruvate oracetate at manometrically detectable rates al-though, as shown earlier, intact cells oxidize
TABLE 2Decarboxylation of oxalacetate by cell-free extracts*
* All flasks contained phosphate buffer 0.005 M(pH 7.3), Mg+ 3.3 X 10-3 M, MnH 1.0 X 104 M,water to 3.2 ml. Gas phase air, temperature 25 C.
t Average of duplicate flasks. Time 30 minutes.
TABLE 3The incorporation of C14 from acetate-S-C4 into
a-ketoglutarate by intact resting cells*
NITALt JNAL
Acetate, pM 158 116a-Ketoglutarate, pM 406 360Acetate specific activity, 434 387m.c/mg C
Acetate total activity, m,uc 1645 1076a-Ketoglutarate specific 0.00 1.06
activity, m,uc/mg Ca-Ketoglutarate total activity, 0.00 22.9mpc* Cells grown 72 hours on rotary shaker in
buffered (pH 6.0) nutrient broth (Difco) plus glu-cose (0.04 M) and MgSO4 (0.001 M).
t Washed cells suspended in buffer (0.017 m,pH 6.0) plus addenda as shown plus water to 40ml, incubated 6 hours on rotary shaker at 25 C.
both. The rapidity of oxidation of citrate in theabsence of added cofactors suggests that suffi-cient triphosphopyridine nucleotide is present inthe preparation, as contrasted with the deficiencyof diphosphopyridine nucleotide evidenced by thedata on fumarate oxidation.The manometric results establish at best only
a presumption as to mechanism. It should benoted that with S. coelicolor, as with most other
cell-free bacterial systems, it was necesary inaerobic experiments to use an artificial hydrogencarrier, methylene blue, to replace the still un-known terminal electron-transfer system. Theremaining experiments to be reported were de-signed to demonstrate as unequivocally as pos-sible the existence of specific reactions. Certain ofthe classical methods for studying metabolic sys-tems proved inapplicable to S. coelicolor; withintact cells, neither sparking experiments normalonate inhibition experiments gave clear-cutresults, and the inability of extracts to oxidizepyruvate and acetate further restricted the pos-sible experimental designs.
The incorporation of acetate--C04 into a-keto-glutarate. It has been shown earlier (Cochrane,1952) that the methyl carbon of acetate can betrapped in succinate although at a low levelonly. In the present work (table 3), exogenous a-ketoglutarate was added to a cell suspensionmetabolizing labeled acetate; the rationale of thismethod is outlined by Ajl (1951).
Utilization of a-ketoglutarate was very slowalthough measurable over the time period used(6 hours).Nevertheless, detectable activity wastrapped in the keto acid, while in the controlsample, made up exactly as the experimental butharvested immediately and purified in parallelwith it, there was none. Of a total of 569 mpclost by acetate in the experimental flask, 23 mpc,or 4 per cent, was recovered in the a-ketoglu-tarate.
Obviously, extracellular a-ketoglutarate is notin equilibrium with the metabolic pool if it isassumed that acetate is oxidized over free a-ketoglutarate. In view of the demonstrationearlier in this paper of the effect of grinding ona-ketoglutarate oxidation, it is believed that theexplanation lies in the relative impermeability ofthe cells to the substrate. We interpret the experi-ment as a qualitative indication of the involve-ment of a-ketoglutarate or a derivative of it inacetate oxidation.The negligible dilution of acetate activity pre-
sumably means that free acetate is not formedfrom a-ketoglutarate. This is in contrast to theresults with glucose (Cochrane, 1952), during theoxidation of which acetate formation can be de-tected both by isotope dilution and by analysis.
The oxidation of ac-ketoglutarate. In the tricar-boxylic acid cycle in animal tissues, a-ketoglu-tarate is oxidatively decarboxylated to succinate,
and the further oxidation of succinate can beblocked by malonate (Stumpf et al., 1947). In thepresence of malonate the respiratory quotientshould approximate 2 and there should be a yieldof one mol of carbon dioxide per mol of substratedisappearing.
Preliminary experiments with cell-free systemsmetabolizing succinate in the presence of malo-nate indicated that the inhibitor is effective, butonly at higher concentrations than usually recom-mended (Pardee and Potter, 1949); a concentra-tion of 0.03 M malonate proved necessary to cause85 per cent inhibition of the oxidation of 0.01 msuccinate.
Using malonate at the level indicated, its effecton the oxidation of a-ketoglutarate (table 4) isfound to be as expected. The respiratory quotientis raised from about 1.4 to about 2.0, and there
* System: Na a-ketoglutarate 0.01 M, phosphatebuffer 0.008 M (pH 7.3), Mg+ 3.3 X 10-3 m,Mn+ 1.0 X 10-4 M, DPN (diphosphopyridinenucleotide) 5.9 X 10-6 M, ATP (adenosine triphos-phate) 1.0 X 10- M, cocarboxylase 8.5 X 10-' M.KOH in center well, time 155 min, temperature30 C, gas phase air.
t Endogenous subtracted.
TABLE 5Metabolic activities of cell-free extracts
OXYGZNPRODUCT
SUBSlTATE(S) ADDENDA* UPTAKAL CompouId Caict Found Ratio
Compound Am Am 420/520?
A. The oxidation of citrate to a-ketoglutarate (KGA)§
None DPN 14 PA - 0.0DL-Malate, 75 pm DPN 86 PA 6.43 5.27 1.37
C. The formation of citrate from malate and acetatell
None YE, ATP, TPP Citrate - 0.41DL-Malate, 60 pm+ acetate, 15 pm YE, ATP, TPP Citrate 2.02
* Na arsenite 6.7 X 10-3 M; DPN (diphosphopyridine nucleotide) 5.9 X 10- m; YE (yeast extract)7.5 mg/ml; ATP (adenosine triphosphate) 7.5 X 10-4 m; TPP (thiamin pyrophosphate) 6.5 X 106 M.
t From oxygen uptake.t Ratio of optical densities at 420 and 520 m,u (for authentic a-ketoglutarate 1.95, for authentic
pyruvate 1.23).§ System: phosphate buffer 0.008 M (pH 7.3); Mg+ 3.3 X 1 i-3M; Mn+ 1 X 10-4 M; methylene blue
1 X 103m; extract 1 ml. Fluid volume 3.2 ml, in Warburg flasks; KOH in center well, gas phase air,temperature 30 C. Time 45 min.
|| System: phosphate buffer 0.006 m (pH 7.3); Mg++ 2.5 X 10-3 m; Mn 1 X 10- M; methylene blue7.5 X 10- m. Volume 4 ml, in test tubes. Temperature 25 C, time 60 min.
is reasonable agreement between carbon dioxide While complete proof of the reaction in ques-evolved and substrate utilized in the presence of tion would involve isolation of the product, suc-malonate. cinic acid, the results presented, particularly the
effect of malonate on the respiratory quotient,seem sufficiently convincing for the present.
The metabolism of citrate. Conversion of citrateto a-ketoglutarate by a cell-free preparation inthe- presence of arsenite and methylene blue isshown in table 5 (A). The agreement betweenoxygen uptake and a-ketoglutarate formation isas close as can be expected. The rate of the reac-tion, if some allowance is made for the effect ofarsenite, is reasonably close to the rate of oxida-tion of citrate by the same preparation (figure2), indicating that in these preparations at leastthe entire metabolism of citrate occurs over a-ketoglutarate.
The metabolism of malate. In the tricarboxylicacid cycle in animal tissues malate is oxidized topyruvate (Wood, 1946). While the sequence isnot of course distinctive, any organism posingthe cycle should convert malate to pyruvate.Since cell-free preparations of S. coelicolor oxidizepyruvate only slowly, it is possible to isolatepyruvate as a product of malate oxidation. Thedata of table 5 (B) indicate that about 83 per centof the oxygen uptake with malate as substratecan be accounted for as pyruvate. No attemptwas made to discriminate between pathways ofmalate oxidation which involve or do not involveoxalacetate as intermediate.
In view of the results described in the nextsection, it is probable that the deficiency ofpyruvate not accounted for represents condensa-tion to citrate, the occurrence of which wouldincrease simultaneously oxygen uptake and de-crease pyruvate recovery.
The formation of citrate. The same cell-freepreparation is shown, in table 5 (C), to carry outthe synthesis of citrate from acetate and malate.Malate was used for convenience; in other re-spects the system used draws upon the demon-stration (Stern and Ochoa, 1949) of a solublecondensing enzyme in animal tissues. Pyruvatecan be substituted for acetate in this system. Theneed for yeast extract and adenosine triphosphatewas not determined; inclusion of diphosphopyri-dine nucleotide was based on its stimulatoryeffect on malate oxidation. Activity of differentpreparations varied widely, from virtually nil toover five times the activity of that shown; in viewof the number of factors involved in this systemand the crude extraction methods used, perhapsthis variability is not surprising.
DISCUSSION
The experiments presented are designed todemonstrate the eistence in S. coelicolor of thereactions of the tricarboxylic acid cycle. Theparticular systems used have been dictated inpart by the nature of the material, particularlythe stability of separated enzymes.The involvement of a-ketoglutarate in acetate
oxidation is indicated by experiments in whichC14 from acetate-2-C'4 is found to be trapped inexogenous a-ketoglutarate. These experiments,by themselves, are not critical because of the verylow level of activity trapped, but the involve-ment of a-ketoglutarate is proved by two otherlines of evidence.
In the first place, citrate is oxidized stoichio-metrically to a-ketoglutarate by cell-free prepara-tions, and the rate is high enough so that it isreasonable to believe that all oxidation of citrateoccurs over a-ketoglutarate. Direct chemicalevidence for the conversion by yeast of citrate toa-ketoglutarate has been reported by Foulkes(1951); Kornberg and Pricer (1951) have shownalso that yeast can carry out the reaction i8o-citrate to ca-ketoglutarate. In the bacteria, Alten-bern and Housewright (1951) have determinedby indirect means that Brucella abortue convertscitrate and cis-aconitate to a-ketoglutarate, theproduct being determined as glutamate aftertransamination or as alanine after a postulatedsequence of reactions involving transamination.
Secondly, the effect of malonate on the oxida-tion of a-ketoglutarate suggests that succinate isits oxidation product. Here the evidence is in-direct, consisting of the demonstration thatmalonate changes the respiratory quotient with a-ketoglutarate as substrate from about 1.4 toabout 2.0. This is taken to mean that the poison,shown experimentally to block the oxidation ofsuccinate at the concentration used, has in effectisolated the oxidative decarboxylation of a-ketoglutarate. The same approach has beenused earlier, e.g., by Stumpf et at. (1947).
Earlier results (Cochrane, 1952) made prob-able the eistence of an oxidative pathway fromsuccinate to pyruvate via fumarate and malatealthough the reductive reactions were the onesdemonstrated. In the present work, we have as-sumed the conversion of succinate to malate onthe basis of the earlier findings, with the addi-tional evidence that diphosphopyridine nucleotide
stimulates succinate and fumarate oxidation bycrude cell-free preparations. The conversion ofmalate to pyruvate by cell-free extracts is shownto occur with good agreement between oxygenuptake and pyruvate recovery.
Finally, the condensation of acetate (or pyru-vate) and a product of malate oxidation, pre-sumably oxalacetate, to citrate is effected by cell-free extracts of S. coelicolor.
While it has been shown that the organismscan decarboxylate oxalacetate, no specific efforthas been made to test for the obligatory involve-ment of oxalacetate in the conversion of malateto pyruvate. However, since malate can supplythe 4-carbon moiety for citrate synthesis, it islikely on grounds of comparative biochemistrythat oxalacetate is in fact an intermediate. Thesame conclusion is suggested by the observationthat either cyanide or glutamate acceleratesmalate oxidation (unpubliAhed data).The entire sequence of reactions postulated
for the tricarboxylic acid cycle in animal cells iscovered by these experiments although severalindividual reactions, e.g., the conversion offumarate to malate and the demonstration ofaconitase, have not been attempted or thoughtworth attempting. While there is of course noevidence as to the quantitative importance ofthe tricarboxylic acid cycle, it seems that theorganism is capable of carrying out all of the re-actions. As in most other microbial systems,aerobic respiration by cell-free extracts requiredan artificial hydrogen carrier, and to that extentthe reactions described are all model systems. Itis to be hoped that further investigations ofterminal respiration properly so-called, i.e., thetransfer of hydrogen to its ultimate acceptor,may in time make more natural systems availablefor study.
It may be noted that there is some evidencein the taxonomically related Mycobacterium thatat least some of the crucial reactions of the cycleoccur (Edson, 1951; Ochoa et al., 1951).
Methodologically, the use of cell-free extractshas proved to be essential, primarily because ofthe low permeability of the celLs to certain keysubstrates. It seems likely that work with otheractinomycetes and with the filamentous fungi willbe facilitated by the use of these methods and forthe same reasons. While there is some danger thattoo great concentration on isolated enzyme sys-
tems will lead to neglect of the organisms as theyexist in nature, the relative simplicity and ab-stractness of these methods should allow morerapid progress in the study of filamentous or-ganisms than has been possible with more complexsystems of growing or resting cells.
ACKNOWLEDGMENT
It is a pleasure to acknowledge the hospitalityof the Brookhaven National Laboratory and ofDr. Martin Gibbs, in whose laboratory the ex-periments with carbon-14 were performed.
5UMMARY
Whole cells oxidize some but not all of thecompounds of the tricarboxylic acid cycle, failingin particular to metabolize citrate and a-keto-glutarate. Cell-free preparations oxidize glucose(with a requirement for adenosine triphosphate),citrate, a-ketoglutarate, succinate, fumarate, andmalate and decarboxylate oxalacetate. Selectedreactions or groups of reactions found to becatalyzed by cell-free extracts include the oxida-tion of citrate to a-ketoglutarate, the conversionof malate to pyruvate, and the condensation ofmalate and acetate (or pyruvate) to citrate.Whole cells incorporate, although at a slow rate,carbon-14 from acetate-2-C4 into a-ketoglu-tarate. The effects of diphosphopyridine nucleo-tide on malate and fumarate oxidation and ofmalonate on the oxidation of a-ketoglutarate arealso consistent with the operation of a tricar-boxylic acid cycle.
It is concluded that Streptomyces coelicolor isable to carry out the reactions of the tricarboxylicacid cycle although no data are as yet availableas to the quantitative importance of this pathwayto the organism.
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