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A reappraisal of the role of the tricarboxylic acid cycle in the respiration of Escherichia coli
A Reappraisal of the Role of the Tricarboxylic Acid Cycle in the Respiration of Escherichiu coli Samuel J. Ajl and Donald T. 0. Wong Prom the Department of Bacteriology, Communicable Diseases Division, Medical Service Graduate School, Washington, D. C. Received
Army
July 20, 1954
Based on experiments utilizing simultaneous adaptation (1) or labeled substrates (2, 3), it appeared that the tricarboxylic acid cycle did not play an important role in the metabolism of acetate-adapted Escherichia c0Zi.l From analysis of residual acids, respiratory COZ , and cell materials, as well as manometric data on oxygen uptake, it was possible to show that during the oxidation of labeled acetate there was incorporation and distribution of labeled carbon into carrier Crdicarboxylic acids and pyruvate. The amount incorporated was that to be expected on the basis of a Thunberg type condensation, whereas there was no comparable incorporation into cr-ketoglutarate (2). However, we reconsidered the observations in the light of the more recent experiments of Saz and Krampita (6), Kaufman et al. (7), and Stadtman and Barker (8) which suggest that activities in externally added carriers may not necessarily reflect the true activities of these intermediates within the cells unless the carrier compounds are in complete equilibrium with the corresponding enzymatically produced intermediates. Since, in our earlier studies, the carboxyl carbons of carrier Cd-dicarboxylic acids were found to be in equilibrium with respiratory CO% , and that of citrate and ar-ketoglutarate were not (2), it became necessary to reinvestigate the mechanism of acetate oxidation in E. coli by large amounts of cells without added carriers. Instead, the intracellular intermediates of acetate oxidation would be isolated and their specific activities studied directly. 1When similar techniques were applied to other organisms, i.e., Micrococcus lysodeikticus (4) and citrate-grown Aerobaeter aerogenes (5), the results were suggestive of the operation of the tricarboxylic acid cycle. 474
TRICARBOXYLIC
ACID
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IN
E. COLI
475
While this work was in progress, detailed and elaborate data of this type were independently reported by Swim and Krampitz (9). The results of their experiments and those to be described here, coupled with the original findings of Barban (10) and those of Swim and Krampitz (11) and Wheat and Ajl (12) that E. coli possesses an active isocitric dehydrogenase and an aconitase, indicates that this organism does indeed respire via the tricarboxylic acid cycle. Further, the failure in our laboratories to demonstrate a condensation of two molecules of acetate to succinate in cell-free extracts of this organism coupled with the recent isotope experiments of Swim and Krampitz (13) with resting cell suspensions under anaerobic conditions makes it doubtful that this type of C&-C2 condensation occurs at all in this organism. METHODS
Escherichia coli (E26) was grown with constant aeration at 30°C. in an acetate medium previously described by Ajl (1). Two procedures were employed with radioactive acetate as substrate. Procedure 1 consisted of incubating methyl-labeled acetate in the presence of one or more unlabeled compounds to which the radioactive intermediate was thought to give rise. At the end of the incubation periods, the reaction mixture was acidified, the cells were centrifuged, the acetate recovered by steam distillation, and the various carriers reisolated by procedures already described in detail elsewhere (14). In procedure 2, methyl-labeled acetate or glucose was metabolized in the absence of carriers by large batches of cells, and attempts were made to isolate the intracellular intermediates of acetate oxidation. This procedure has been described in detail by Glover et al. (15) and employed in this investigation as modified by Santer and Ajl (14). In aerobic experiments, air was constantly passed through the reaction mixture in loo-ml. test tubes in order to maintain adequate aerobiosis. Cell-free extracts were prepared by grinding the cells with either alumina 303 according to the procedure of McIlwain (16) or with ground glass according to the method of Halnitsky et al. (17). The isolation procedures for succinate, fumarate, malate, and cu-ketoglutarate were described in detail elsewhere (14). a-Betoglutarate was determined quantitatively by the method of Friedemann and Haugen (18), separated chromatographically by a modification of the method of Seligson and Shapiro (19), and degraded with ceric sulfate (20); citrate concentration was determined by the method of Natelson et al. (21) and degraded with ceric sulfate (20). Succinate was estimated and degraded by the succinoxidase method. Methods for the determination of carbon-14 were as previously described (2). 2 The experiments ington University
of Dr. Barban Medical School,
were performed St. Louis, MO.
in our
laboratories
at Wash-
476
8. J. AJL AND D. T. 0. WONG EXPERIMENTAL
Oxidation of Labeled Acetate by Resting Cell Suspensionsof E. coli in the Presenceof Externally Added Carriers For comparative purposes, initial experiments involved the oxidation of a radioactive substrate in the presence of postulated intermediates and determining whether any activity became incorporated in the latter. Labeled acetate was, therefore, oxidized in the presence of unlabeled succinate, cu-ketoglutarate, and citrate. Although during the course of the experiment both succinate and ar-ketoglutarate were available to the bacteria, as evidenced by both oxidative metabolism and substrate disappearance, acetate carbon was recovered primarily in succinate. Citrate was neither oxidized nor did it become radioactive. The results are summarized in Table I and are essentially similar to those previously published by Ajl and Kamen (2) and Swim and Krampitz (9). From the data given in Table I, it was calculated that out of the 22,475 counts/min. that were found in succinate, 13,361 counts/min. were contained in the methylene and 9,052 counts/min. in the carboxyl carbons. Thus, it appeared that sufficient cycling had occurred to fix appreciable methyl carbon of acetate in succinyl carboxyl. From the total activity of carboxyl carbon and the concentration of residual succinate, it was possible to calculate the specific activity of each carboxyl carbon of the C&dicarboxylic acid. A value of 28 counts/min./PM single carboxyl was obtained. Examination of the carbonate data revealed that the average specific activity of the CO2 evolved was 34 counts/min./PM. Thus, these findings are in accord with the notion that succinyl carboxyl is in equilibrium with evolved CO2 , as might be expected on the basis of a cyclic mechanism operating by way of a C2-C2 condensation. Subsequent experimental data, however, did not bear out this contention. Oxidation of Labeled Acetate by Lyophilized and Sulfuric Acid-Dried E. coli in the Presenceof Externally Added Carriers The observation that citrate did not become radioactive during the course of the oxidation of radioactive acetate was not surprising since it is well known that untreated resting cells of E. coli do not metabolize this C&ricarboxylic acid. In view, however, of the recent findings in our laboratories (12) and by other investigators (22), that aluminaground cell-free extracts and dried preparations of this organism do
TRICARBOXYLIC
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TABLE
I
IN
E.
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COLI
Quantitative Observations on the Oxidation of Labeled Acetate by Escherichia in the Presence of Externally Added Succinate, a-Ketoglutarate and Citrate The complete system contained 2 ml. of a 10% suspension of freshly salts of substrates as indicated; and ml., temp. 33°C. Time of incubation,
I
Product
4mount d
ml. of 0.2 M phosphate buffer of pH 7.0; 1.5 harvested (acetate-grown) E. coli; sodium NaOH in the center well. Total volume 12 4 hr. c “4 conten colunts/min.
186 207 207 207
189,240
Initial conditions
Acetate Succinate a-Ketoglutarate Citrate Carbonate
224 161
124,000 22,475 13,361
Final conditions
Acetate Succinate Methylene carbons Carboxyl carbons a-Ketoglutarate Citrate Cells (66 mg. dry weight) Carbonate
42
0 0 0 0
186 94 3,618 2,836
t
Specific
Activity
counts/min/pM
9,052 157 205
coli
Manometric data, oxygen uptake; pl. Exptl.
1017 0 0 0 0
Endogenous
6057
2202
6057 - 2202 3855Pl. Calculated from substrates disappeared 6068 pl.
554 139.5 41 28 1.1
0.45
3 4(average specific tivity)
ac-
attack citric acid, it was of some interest to learn whether the oxidation of labeled acetate by variously dried preparations of E. wli would result in the incorporation of acetate carbon into the G-acid. Typical results are exhibited in Table II. All five experiments described in Table II were performed simultaneously with equivalent cell concentrations so that direct comparisons among them is permissible. The data obtained with both sulfuric aciddried and lyophilized preparations are strikingly different from those obtained with untreated resting cells. Qualitatively, the salient results with the dried cells were as follows: (a) the marked disappearance of
478
s.
J. AJL
AND
D.
T.
0. WONG
TABLE II of Radioactive Acetate in the Presence of Tricarboxylic Acid Cycle Intermediates by Sulfuric Acid-Dried and Lyophilized Escherichia coli
Oxidation
Each 125.ml. Warburg reaction vessel contained the indicated amount of substrate, 2 ml. of 0.2 il4 phosphate buffer, pH 7.0, 100 &kf of sodium 2-P-acetate containing approximately 2 X lo6 cts./min., 200 mg. of either sulfuric acid-dried, lyophilized or wet cells and 3 ml. of NaOH in center well. Temperature 33°C. Time of incubation 4 hours. ,__. z=z Final Amount specific Average “fi:r’
Cell
preparation
1 Sulfuric 2
Fraction
Citrate Succinate” Evolved CO,
acidor-Ketoglutardried cells ate Succinate” Evolved COS
-3 Lyophi4
5
lized cells
Untreated resting cells
Citrate Succinate” Evolved CO, a-Ketoglutarate Succinate” Evolved CO% _~~--__~____ Citrate Succinate” a-Ketoglutarate
Cl4 content Initial _______-
Final
PM
rM
FiIlal specific activity
rctivity of
specific activity
‘ arboxyl
of COz
carbon .-
counlslmin.
counts/ counts/
mi?&M
61,590 714 477,200 6,723 776,900 1,337 55,410
265
53
0 63 0 542 __-_____ 300 113 0 23 0 1108 ----300 197
419,400 896,150
6,671 1,654
1,158
43,250 383 208,380 6,830 2,433,OOO 2,196
64 1,196
23 603
119 1,008 _-
668
---
.-
827
.-
_-
22,670
CL
n rin.lpM
300 86.2 0 70.9 0 581 --~~ 300 209
0 0
e7 mlved
115
28
101,640 4,447 2,104,500 3,490
1,112
1,098 ----
1,745 .-
300 0 300
296 2 253
360 1.2 13,480 8,380 720 3
mFormed during the course of reaction. The amount of enzymatically succinate from citrate by untreated resting cells is practically nil.
formed
both citric and tu-ketoglutaric acids and the incorporation of acetateCl4 into the Cg- and C&-acids, and (b) the enzymatic formation and accumulation of succinate from both citrate and cr-ketoglutarate. Quantitatively, the major observation was the non-equilibration of respira-
TRICARBOXYLIC
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IN
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479
tory COZ with carboxyl carbon of either citrate or a-ketoglutarate. Untreated resting cells gave similar results to those described in Table I. Oxidation of Labeled Acetate by Whole Cells in the Absence of Added Carriers From the data thus far presented it would appear that citrate does not become active during the oxidation of radioactive acetate by untreated resting cells simply because of permeability considerations. The reason for the incorporation of acetate carbon into ol-ketoglutarate by dried cells is not as yet apparent since both untreated and dried suspensions of E. coli metabolize this C&-keto acid. Despite the observations with the dried cell preparations, it still could not be assumed that both citrate and a-ketoglutarate participate quantitatively in acetate oxidation. This is because the specific activity of the respiratory CO2 is only in equilibrium with succinyl carboxyl but is manyfold higher than that of carboxyl carbon of either citrate or cr-ketoglutarate. Unequivocal proof for the participation of the latter two acids in acetate oxidation mandates the demonstration of equivalence between respiratory CO2 and carboxyl carbon of the latter two acids. This can indeed be demonstrated, at least as far as citrate is concerned, by studying the oxidation of labeled acetate in the presence of large amounts of cells without added carriers, as was done by Swim and Krampitz (9) with E. coli and by Santer and Ajl in the case of Pasteurella pestis (14). Accordingly, resting cells of E. coli were allowed to oxidize methyllabeled acetate (without added carriers) for varying periods of time, after which the cells were disrupted and the intracellular Krebs cycle intermediates separated chromatographically and their concentrations determined quantitatively. The results are exhibited in Table III. Several observations deserve comment: (a) Citric acid can be extracted from E. coli and, during the oxidation of labeled acetate, intracellular citrate becomes active; (b) citric acid has a higher specific activity than succinate at the initial stages and a lower specific activity toward the end of the experiment; (c) the intracellular concentration of citrate is between 200 and 500 times lower than succinate; and (d) although methyl-labeled acetate was oxidized, the respiratory COz becomes active and its specij’k activity is indeed equivalent to the speciJic activity of the individual carboxyls of both sue&ate and citrate. a-Ketoglutarate could not be isolated from as much as 20-40 g. wet weight of E. coli. It is to be emphasized, however, that some activity
480
S.
Equivalence
of During
Respiratory Oxidation
J.
AJL
AND
D.
T.
TABLE III CO, and Carboxyl
of Methyl-Labeled
0.
WONG
Carbon of Succinate by Escherichia
Acetate
Citrate
and
coli
Total volume of reactants, 100 ml. Reaction flask contained 50 g. (wet weight) of E. coli; 2 ml. of 0.2 M phosphate buffer, pH 7.2; 1 m&4 radioactive acetic acid containing 17.5 X 10’ counts/min.; and distilled water to volume. Temperature, 25°C. Aerobic. Time interval min. 90
180 360
Fraction
Succinate Citrate Succinate Citrate Succinate Citrate
Intracellular concentration PM
537.5 0.75 278.0 1.07 240.0 0.78
Specific activity counts/min./&f
8,020 14,510 14,030 11,530 17,880 15,700
Specific activity er single car B oxyl carbon counts/min./pM
144 105 304 371 -
Specilk activity of respiratory COz counts/min.fpM
143 344 773
was always present in the band corresponding to the Cb-keto acid on a paper chromatogram. However, when very large amounts of cells were analyzed for a-ketoglutarate, minute quantities of the free acid could be detected. Oxidation of Labeled Acetate by Glucose-Grown E. coli in the Absence of Added Carriers An interesting observation was made concerning a-ketoglutarate during the oxidation of labeled acetate by glucose-grown E. coli. As was stated earlier, this C&keto acid never becomes appreciably active when untreated acetate-grown organisms are allowed to metabolize acetateCY4. However, when glucose-grown cells are exposed to radioactive acetic acid, intracellular a-ketoglutarate becomes highly active, and this activity often equals that associated with succinic acid (Table IV). This observation strengthens the supposition that E. coli can metabolize acetate via a-ketoglutarate. Of greater interest is the possibility that the enzymes of the Krebs cycle are to some extent under adaptive control and that different growth substrates will yield cells with different enzymic composition. Experiments with Glass-Ground Cell-Free Extracts of E. coli Although experiments with intracellular intermediates provided ample evidence for the participation of citrate in the oxidation of acetate, it was nevertheless desirable to learn whether cell-free extracts of E.
TRICARBOXYLIC
ACID
CYCLE
TABLE Acetic
Acid
Oxidation
IN
E.
481
COLI
IV
by Glucose-
and Acetate-Grown
Cells
Total volume of reactants, 20 ml. Each flask contained 26 g. (wet cells; 3 ml. of 0.2 M phosphate buffer, pH 7.4; and 5 g. of sodium acetate 1 X 10’ counts/min. Time, l%j hr. Aerobic. Room temperature. Distribution
of activity
in intracellular
CiS-AC-
“3”.
10 2a
Preparation
Glucose-grown Acetate-grown
onitate
Succinate counts/ counts/ min. min.
cells cells
1187 2185
40,440 ,91,&f
Fumarate
Malate
counts/ counts/ min.
min.
4,280 5,310
4,465 3,215
weight) containing
of
intermediates a-Ketoglutarate Citrate counts/ counts/ min. min.
61,440 661
11,650 4,320
a Experiments 1 and 2 were carried out at different times and are not to be compared with each other. Rather, the ratios of activities of succinate to a-ketoglutarate in each individual experiment are the significant observations in connection with the data presented in this table.
coli would bring about a condensation of two acetate molecules to succinate. No detectable succinate formed in the presence of either acetate alone or acetate and diphosphopyridine or triphosphopyridine nucleotide, adenosine triphosphate, coenzyme A or flavin adenine dinucleotide. Diphosphopyridine nucleotide enhances the reduction of methylene blue in the presence of acetate, but the reduction of the dye does not result in succinate formation from the added acetate. It, therefore, seems reasonable to conclude that E. wli does not contain an enzyme condensing acetate to succinate. Participation
of the Tricarboxylic
Acid Cycle in Glucose Oxidation
by
E. coli It was of interest to determine whether the tricarboxylic acid cycle participates in glucose oxidation by E. wli. Accordingly, a large quantity of resting cells was added to randomly labeled glucose and, at the end of incubation, the intracellular Krebs cycle intermediates were isolated and their activities determined. Both the di- and tricarboxylic acids became active, indicating their qualitative participation in glucose oxidation. Participation
of the Tricarboxylic Acid Cycle in Acetate Metabolism Growing Cells of E. coli
by
The participation of the tricarboxylic acid cycle intermediates in growing cells is evident from the data presented in Table V. Ten grams (wet weight) of cells was inoculated into 16 1. of a complete acetate
482
S.
J.
AJL
AND
D.
TABLE Participation
of the Tricarboxylic Growing Cells
Intermediate
Citrate a-Ketoglutarate Succinate Malate” Fumarate” cis-AconitateO (1 The
concentration
T.
of these
WONG
V
Acid Cycle of Escherichia
Concentration dJ
Could
0.
0.291 not be detected 143.3 intermediates
in Acetate coli
Metabolism
by
C” content counls/min.
Specific activity counlslmin. j&f
4,563 16,500 61,675 3,688 4,187 5,750
15,573
was not
430
determined.
medium to which were added 500 pM of acetate-2-Cl4 containing approximately 1 X 10’ counts/mm. After a 12-hr. period of incubation with constant aeration, 40 g. of cells was obtained. These were broken up, and the intracellular Krebs cycle intermediates were isolated and studied. It is quite evident from the data shown in Table V that all intermediates became radioactive, including cr-ketoglutarate.
DISCUSSION The experiments described in this, and in a recently published communication from our laboratories (12), coupled with those of Krampitz and co-workers (9, ll), leave no doubt that E. coli not only possesses all of the component enzymes of the tricarboxylic acid cycle but that it oxidizes acetate via this same mechanism. The complete equivalence between respiratory COZ and carboxyl carbons of both intracellular citrate and succinate during the oxidation of methyl-labeled acetate constitutes the major evidence for this assumption. The finding that a-ketoglutarate becomes highly active when radioactive acetic acid is oxidized by dried cells adds considerable weight to the hypothesis that this keto acid participates in the oxidization of the G-fatty acid. The failure to demonstrate a back-to-back condensation of two molecules of acetate to succinate in cell-free extracts of E. coli minimizes the possibility that a dicarboxylic acid cycle exists in this bacterium. It now appears that the earlier published data [recently summarized by Ajl (23)] dealing with (a) the equilibration of respiratory CO2 with succinyl carboxyl, (5) the failure of carrier citrate and cY-ketoglutarate to trap acetate carbon, and (c) quantitative data on 02 uptake and substrate disappearance during the oxidation of labeled acetate by nonprolifer.at-
TRICARBOXYLIC
ACID
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IN
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483
ing cells were not sufficient to eliminate the tricarboxylic acid cycle as a terminal respiratory pathway in E. coli. On reinvestigating this problem by studying the intracellular intermediates of acetate oxidation (and not the externally added carriers), it becomes quite clear that citrate carboxyl, for example, not only equilibrates with respiratory COz but that the specific activity of the G-acid precurses succinate during the early stages of the experiment and falls below that of the Cd-acid toward the end of the experiment as the tricarboxylic acid cycle mandates. It should be noted that although carrier citrate as well as a-ketoglutarate becomes active during acetate-Cl4 oxidation by dried cells, these acids are not in equilibrium with respiratory COZ . The failure to demonstrate equivalance between respiratory COZ and carboxyl carbon of cY-ketoglutarate under any of our conditions is perplexing. Even when as many as 40 g. of cells was broken up, with the methodology employed by the authors, it was impossible to determine with any degree of accuracy any detectable intracellular a-ketoglutarate. The evidence for the participation of this keto acid in acetate oxidation comes, therefore, from the results of Swim and Krampitz (9) who succeeded in demonstrating equivalance between respiratory CO2 and intracellular a-ketoglutaric acid. Their findings, coupled with our observation that the band corresponding to the Cr,-keto acid on a paper chromatogram always contained some activity (making the specific activity of the acid extremely high and a potential precursor of succinate), does not justify the elimination of this keto acid as an intermediate in acetate oxidation by E. coli. The occurrence of the enzymes aconitase and isocitric dehydrogenase (N-12) adds considerable weight to this supposition, since these when combined convert quantitatively citric acid to c\l-ketoglutaric acid. The possibility exists that the enzyme which splits citric acid into Cd and Cz units, recently described by Ajl and co-workers (12, 24) and others (25, 26) or the enzymes which split cis-aconitate or isocitrate to succinate and glyoxylate recently found to be present in E. coli in our laboratories (27) and reported by Campbell et al. (28) to be present in Pseudomonas aemginosa (functioning with cis-aconitate) operates in this bacterium to such an extent that the bulk of tricarboxylic acid breakdown does not proceed via a-ketoglutarate but to succinate and acetate or glyoxylate directly. Although the tricarboxylic acid cycle has now been shown to occur in a number of microorganisms, it would be premature to conclude that it exists and operates in all of them.
484
S. J. AJL
AND
D. T.
0.
WONG
&JMMARY
By studying
the intracellular intermediates of acetate oxidation in equilibration between respiratory CO2 and carboxyl carbon of both citrate and succinate has been demonstrated. Dried preparations incorporate significant amounts of acetate carbon into all Krebs cycle intermediates. These data and those of Krampitz and co-workers, plus the finding that this organism contains a potent isocitric dehydrogenase and an aconitase, strongly suggest that E. wli does indeed respire via the tricarboxylic acid cycle. The failure to date to demonstrate an acetate-to-acetate condensing enzyme in cell-free extracts leaves much doubt whether this bacterium oxidizes acetate via an abridged type of cyclic mechanism, as the results with nonproliferating, resting cells previously suggested.
Escherichia coti, complete
REFERENCES 1. AJL, S. J., J. Bacterial. 69,499 (1950). 2. AJL, S. J., AND KAMEN, M. D., J. Biol. Chem. 189,845 (1951). 3. AJL, S. J., J. Gen. Physiol. 34,785 (1951). 4. AJL, S. J., KAMEN, M. D., RANSON, S. L., AND WONO, D. T. O., J. Biol. Chem. 189, 859 (1951). 5. AJL, S. J., AND WONO, D. T. O., J. Bacterial. 61,379 (1951). 6. SAZ, H. J., AND KRAMPITZ, L. O., J. Bacterial. 67, 409 (1954). 7. KAUFMAN, S., KOREES, S., AND DEL CAMPILLO, A., J. Biol. Chem. 192, 301 (1951). 8. STADTMAN, E. R., AND BARKER, H. A., J. Biol. Chem. 180,1169 (1949). 9. SWIM, H. E., AND KRAMPITZ, L. O., J. Bacterial. 67, 419 (1954). 10. BARBAN, S., “Enzymatic Mechanisms of Carbon Dioxide Assimilation in Bacteria.” Ph. D. dissertation, Washington University, 1953. 11. SWIM, H. E., AND KRAMPITZ, L. O., Federation Proc. 11,296 (1952). 12. WHEAT, R. W., AND AJL, S. J., Arch. Biochem. and Biophys. 49,7 (1954). 13. SWIM, H. E., AND KRAMPITZ, L. O., J. Bacterial. 67,426 (1954). 14. SANTER, M., AND AJL, S. J., J. Bacterial. 67,379 (1954). 15. GLOVER, J., KAMEN, M. D., AND VAN GENDEREN, H., Arch. Biochem. and Biophys. 36, 384 (1952). 16. MCILWAIN, H. J., J. Gen. Physiol. 2, ‘2% (1948). 17. KALNITSKY, G., UTTER, M. F., AND WERKMAN, C. H., J. Bacterial. 49, 595 (1945). 18. FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 19. SELIGSON, D., AND SHAPIRO, B., Anal. Chem. 24,754 (1952). 20. AJL, S. J., WONG, D. T. O., AND HERSEY, D. F., .7. Am. Chem. Sot. 74, 553 (1952). 21. NATELSON, S., PINCUS, J. B., AND LUGOVOY, J. K., f. BioE. Chem. 176, 745 (1948).
TRICARBOXYLIC
22. 23. 24. 26. 26. 27. 28.
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CYCLE
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COLI
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LARA, F. J. S., AND STOKES, J. L., J. Bacterial. 63, 415 (1952). AJL, S. J., Bacterial. Revs. 16, 211 (1951). WHEAT, R. W., WONG, D. T. O., AND AJL, S. J., J. Bacterial. 68, 19 (1954). GILLESPIE, D. C., AND GUNSALUS, I. C., Bacterial. Proc. 80 (1953). DAGLEY, S., AND DAWES, E. A., Nature 173, 345 (1953). WONG, D. T. O., AND AJL, S. J., to be published. CAMPBELL, J. J. R., SMITH, R. A., AND EAGLES, B. A., Biochim et. Biophys. Acta 11,594 (1953).
Army
July 20, 1954
Based on experiments utilizing simultaneous adaptation (1) or labeled substrates (2, 3), it appeared that the tricarboxylic acid cycle did not play an important role in the metabolism of acetate-adapted Escherichia c0Zi.l From analysis of residual acids, respiratory COZ , and cell materials, as well as manometric data on oxygen uptake, it was possible to show that during the oxidation of labeled acetate there was incorporation and distribution of labeled carbon into carrier Crdicarboxylic acids and pyruvate. The amount incorporated was that to be expected on the basis of a Thunberg type condensation, whereas there was no comparable incorporation into cr-ketoglutarate (2). However, we reconsidered the observations in the light of the more recent experiments of Saz and Krampita (6), Kaufman et al. (7), and Stadtman and Barker (8) which suggest that activities in externally added carriers may not necessarily reflect the true activities of these intermediates within the cells unless the carrier compounds are in complete equilibrium with the corresponding enzymatically produced intermediates. Since, in our earlier studies, the carboxyl carbons of carrier Cd-dicarboxylic acids were found to be in equilibrium with respiratory CO% , and that of citrate and ar-ketoglutarate were not (2), it became necessary to reinvestigate the mechanism of acetate oxidation in E. coli by large amounts of cells without added carriers. Instead, the intracellular intermediates of acetate oxidation would be isolated and their specific activities studied directly. 1When similar techniques were applied to other organisms, i.e., Micrococcus lysodeikticus (4) and citrate-grown Aerobaeter aerogenes (5), the results were suggestive of the operation of the tricarboxylic acid cycle. 474
TRICARBOXYLIC
ACID
CYCLE
IN
E. COLI
475
While this work was in progress, detailed and elaborate data of this type were independently reported by Swim and Krampitz (9). The results of their experiments and those to be described here, coupled with the original findings of Barban (10) and those of Swim and Krampitz (11) and Wheat and Ajl (12) that E. coli possesses an active isocitric dehydrogenase and an aconitase, indicates that this organism does indeed respire via the tricarboxylic acid cycle. Further, the failure in our laboratories to demonstrate a condensation of two molecules of acetate to succinate in cell-free extracts of this organism coupled with the recent isotope experiments of Swim and Krampitz (13) with resting cell suspensions under anaerobic conditions makes it doubtful that this type of C&-C2 condensation occurs at all in this organism. METHODS
Escherichia coli (E26) was grown with constant aeration at 30°C. in an acetate medium previously described by Ajl (1). Two procedures were employed with radioactive acetate as substrate. Procedure 1 consisted of incubating methyl-labeled acetate in the presence of one or more unlabeled compounds to which the radioactive intermediate was thought to give rise. At the end of the incubation periods, the reaction mixture was acidified, the cells were centrifuged, the acetate recovered by steam distillation, and the various carriers reisolated by procedures already described in detail elsewhere (14). In procedure 2, methyl-labeled acetate or glucose was metabolized in the absence of carriers by large batches of cells, and attempts were made to isolate the intracellular intermediates of acetate oxidation. This procedure has been described in detail by Glover et al. (15) and employed in this investigation as modified by Santer and Ajl (14). In aerobic experiments, air was constantly passed through the reaction mixture in loo-ml. test tubes in order to maintain adequate aerobiosis. Cell-free extracts were prepared by grinding the cells with either alumina 303 according to the procedure of McIlwain (16) or with ground glass according to the method of Halnitsky et al. (17). The isolation procedures for succinate, fumarate, malate, and cu-ketoglutarate were described in detail elsewhere (14). a-Betoglutarate was determined quantitatively by the method of Friedemann and Haugen (18), separated chromatographically by a modification of the method of Seligson and Shapiro (19), and degraded with ceric sulfate (20); citrate concentration was determined by the method of Natelson et al. (21) and degraded with ceric sulfate (20). Succinate was estimated and degraded by the succinoxidase method. Methods for the determination of carbon-14 were as previously described (2). 2 The experiments ington University
of Dr. Barban Medical School,
were performed St. Louis, MO.
in our
laboratories
at Wash-
476
8. J. AJL AND D. T. 0. WONG EXPERIMENTAL
Oxidation of Labeled Acetate by Resting Cell Suspensionsof E. coli in the Presenceof Externally Added Carriers For comparative purposes, initial experiments involved the oxidation of a radioactive substrate in the presence of postulated intermediates and determining whether any activity became incorporated in the latter. Labeled acetate was, therefore, oxidized in the presence of unlabeled succinate, cu-ketoglutarate, and citrate. Although during the course of the experiment both succinate and ar-ketoglutarate were available to the bacteria, as evidenced by both oxidative metabolism and substrate disappearance, acetate carbon was recovered primarily in succinate. Citrate was neither oxidized nor did it become radioactive. The results are summarized in Table I and are essentially similar to those previously published by Ajl and Kamen (2) and Swim and Krampitz (9). From the data given in Table I, it was calculated that out of the 22,475 counts/min. that were found in succinate, 13,361 counts/min. were contained in the methylene and 9,052 counts/min. in the carboxyl carbons. Thus, it appeared that sufficient cycling had occurred to fix appreciable methyl carbon of acetate in succinyl carboxyl. From the total activity of carboxyl carbon and the concentration of residual succinate, it was possible to calculate the specific activity of each carboxyl carbon of the C&dicarboxylic acid. A value of 28 counts/min./PM single carboxyl was obtained. Examination of the carbonate data revealed that the average specific activity of the CO2 evolved was 34 counts/min./PM. Thus, these findings are in accord with the notion that succinyl carboxyl is in equilibrium with evolved CO2 , as might be expected on the basis of a cyclic mechanism operating by way of a C2-C2 condensation. Subsequent experimental data, however, did not bear out this contention. Oxidation of Labeled Acetate by Lyophilized and Sulfuric Acid-Dried E. coli in the Presenceof Externally Added Carriers The observation that citrate did not become radioactive during the course of the oxidation of radioactive acetate was not surprising since it is well known that untreated resting cells of E. coli do not metabolize this C&ricarboxylic acid. In view, however, of the recent findings in our laboratories (12) and by other investigators (22), that aluminaground cell-free extracts and dried preparations of this organism do
TRICARBOXYLIC
ACID
CYCLE
TABLE
I
IN
E.
477
COLI
Quantitative Observations on the Oxidation of Labeled Acetate by Escherichia in the Presence of Externally Added Succinate, a-Ketoglutarate and Citrate The complete system contained 2 ml. of a 10% suspension of freshly salts of substrates as indicated; and ml., temp. 33°C. Time of incubation,
I
Product
4mount d
ml. of 0.2 M phosphate buffer of pH 7.0; 1.5 harvested (acetate-grown) E. coli; sodium NaOH in the center well. Total volume 12 4 hr. c “4 conten colunts/min.
186 207 207 207
189,240
Initial conditions
Acetate Succinate a-Ketoglutarate Citrate Carbonate
224 161
124,000 22,475 13,361
Final conditions
Acetate Succinate Methylene carbons Carboxyl carbons a-Ketoglutarate Citrate Cells (66 mg. dry weight) Carbonate
42
0 0 0 0
186 94 3,618 2,836
t
Specific
Activity
counts/min/pM
9,052 157 205
coli
Manometric data, oxygen uptake; pl. Exptl.
1017 0 0 0 0
Endogenous
6057
2202
6057 - 2202 3855Pl. Calculated from substrates disappeared 6068 pl.
554 139.5 41 28 1.1
0.45
3 4(average specific tivity)
ac-
attack citric acid, it was of some interest to learn whether the oxidation of labeled acetate by variously dried preparations of E. wli would result in the incorporation of acetate carbon into the G-acid. Typical results are exhibited in Table II. All five experiments described in Table II were performed simultaneously with equivalent cell concentrations so that direct comparisons among them is permissible. The data obtained with both sulfuric aciddried and lyophilized preparations are strikingly different from those obtained with untreated resting cells. Qualitatively, the salient results with the dried cells were as follows: (a) the marked disappearance of
478
s.
J. AJL
AND
D.
T.
0. WONG
TABLE II of Radioactive Acetate in the Presence of Tricarboxylic Acid Cycle Intermediates by Sulfuric Acid-Dried and Lyophilized Escherichia coli
Oxidation
Each 125.ml. Warburg reaction vessel contained the indicated amount of substrate, 2 ml. of 0.2 il4 phosphate buffer, pH 7.0, 100 &kf of sodium 2-P-acetate containing approximately 2 X lo6 cts./min., 200 mg. of either sulfuric acid-dried, lyophilized or wet cells and 3 ml. of NaOH in center well. Temperature 33°C. Time of incubation 4 hours. ,__. z=z Final Amount specific Average “fi:r’
Cell
preparation
1 Sulfuric 2
Fraction
Citrate Succinate” Evolved CO,
acidor-Ketoglutardried cells ate Succinate” Evolved COS
-3 Lyophi4
5
lized cells
Untreated resting cells
Citrate Succinate” Evolved CO, a-Ketoglutarate Succinate” Evolved CO% _~~--__~____ Citrate Succinate” a-Ketoglutarate
Cl4 content Initial _______-
Final
PM
rM
FiIlal specific activity
rctivity of
specific activity
‘ arboxyl
of COz
carbon .-
counlslmin.
counts/ counts/
mi?&M
61,590 714 477,200 6,723 776,900 1,337 55,410
265
53
0 63 0 542 __-_____ 300 113 0 23 0 1108 ----300 197
419,400 896,150
6,671 1,654
1,158
43,250 383 208,380 6,830 2,433,OOO 2,196
64 1,196
23 603
119 1,008 _-
668
---
.-
827
.-
_-
22,670
CL
n rin.lpM
300 86.2 0 70.9 0 581 --~~ 300 209
0 0
e7 mlved
115
28
101,640 4,447 2,104,500 3,490
1,112
1,098 ----
1,745 .-
300 0 300
296 2 253
360 1.2 13,480 8,380 720 3
mFormed during the course of reaction. The amount of enzymatically succinate from citrate by untreated resting cells is practically nil.
formed
both citric and tu-ketoglutaric acids and the incorporation of acetateCl4 into the Cg- and C&-acids, and (b) the enzymatic formation and accumulation of succinate from both citrate and cr-ketoglutarate. Quantitatively, the major observation was the non-equilibration of respira-
TRICARBOXYLIC
ACID
CYCLE
IN
E.
COLI
479
tory COZ with carboxyl carbon of either citrate or a-ketoglutarate. Untreated resting cells gave similar results to those described in Table I. Oxidation of Labeled Acetate by Whole Cells in the Absence of Added Carriers From the data thus far presented it would appear that citrate does not become active during the oxidation of radioactive acetate by untreated resting cells simply because of permeability considerations. The reason for the incorporation of acetate carbon into ol-ketoglutarate by dried cells is not as yet apparent since both untreated and dried suspensions of E. coli metabolize this C&-keto acid. Despite the observations with the dried cell preparations, it still could not be assumed that both citrate and a-ketoglutarate participate quantitatively in acetate oxidation. This is because the specific activity of the respiratory CO2 is only in equilibrium with succinyl carboxyl but is manyfold higher than that of carboxyl carbon of either citrate or cr-ketoglutarate. Unequivocal proof for the participation of the latter two acids in acetate oxidation mandates the demonstration of equivalence between respiratory CO2 and carboxyl carbon of the latter two acids. This can indeed be demonstrated, at least as far as citrate is concerned, by studying the oxidation of labeled acetate in the presence of large amounts of cells without added carriers, as was done by Swim and Krampitz (9) with E. coli and by Santer and Ajl in the case of Pasteurella pestis (14). Accordingly, resting cells of E. coli were allowed to oxidize methyllabeled acetate (without added carriers) for varying periods of time, after which the cells were disrupted and the intracellular Krebs cycle intermediates separated chromatographically and their concentrations determined quantitatively. The results are exhibited in Table III. Several observations deserve comment: (a) Citric acid can be extracted from E. coli and, during the oxidation of labeled acetate, intracellular citrate becomes active; (b) citric acid has a higher specific activity than succinate at the initial stages and a lower specific activity toward the end of the experiment; (c) the intracellular concentration of citrate is between 200 and 500 times lower than succinate; and (d) although methyl-labeled acetate was oxidized, the respiratory COz becomes active and its specij’k activity is indeed equivalent to the speciJic activity of the individual carboxyls of both sue&ate and citrate. a-Ketoglutarate could not be isolated from as much as 20-40 g. wet weight of E. coli. It is to be emphasized, however, that some activity
480
S.
Equivalence
of During
Respiratory Oxidation
J.
AJL
AND
D.
T.
TABLE III CO, and Carboxyl
of Methyl-Labeled
0.
WONG
Carbon of Succinate by Escherichia
Acetate
Citrate
and
coli
Total volume of reactants, 100 ml. Reaction flask contained 50 g. (wet weight) of E. coli; 2 ml. of 0.2 M phosphate buffer, pH 7.2; 1 m&4 radioactive acetic acid containing 17.5 X 10’ counts/min.; and distilled water to volume. Temperature, 25°C. Aerobic. Time interval min. 90
180 360
Fraction
Succinate Citrate Succinate Citrate Succinate Citrate
Intracellular concentration PM
537.5 0.75 278.0 1.07 240.0 0.78
Specific activity counts/min./&f
8,020 14,510 14,030 11,530 17,880 15,700
Specific activity er single car B oxyl carbon counts/min./pM
144 105 304 371 -
Specilk activity of respiratory COz counts/min.fpM
143 344 773
was always present in the band corresponding to the Cb-keto acid on a paper chromatogram. However, when very large amounts of cells were analyzed for a-ketoglutarate, minute quantities of the free acid could be detected. Oxidation of Labeled Acetate by Glucose-Grown E. coli in the Absence of Added Carriers An interesting observation was made concerning a-ketoglutarate during the oxidation of labeled acetate by glucose-grown E. coli. As was stated earlier, this C&keto acid never becomes appreciably active when untreated acetate-grown organisms are allowed to metabolize acetateCY4. However, when glucose-grown cells are exposed to radioactive acetic acid, intracellular a-ketoglutarate becomes highly active, and this activity often equals that associated with succinic acid (Table IV). This observation strengthens the supposition that E. coli can metabolize acetate via a-ketoglutarate. Of greater interest is the possibility that the enzymes of the Krebs cycle are to some extent under adaptive control and that different growth substrates will yield cells with different enzymic composition. Experiments with Glass-Ground Cell-Free Extracts of E. coli Although experiments with intracellular intermediates provided ample evidence for the participation of citrate in the oxidation of acetate, it was nevertheless desirable to learn whether cell-free extracts of E.
TRICARBOXYLIC
ACID
CYCLE
TABLE Acetic
Acid
Oxidation
IN
E.
481
COLI
IV
by Glucose-
and Acetate-Grown
Cells
Total volume of reactants, 20 ml. Each flask contained 26 g. (wet cells; 3 ml. of 0.2 M phosphate buffer, pH 7.4; and 5 g. of sodium acetate 1 X 10’ counts/min. Time, l%j hr. Aerobic. Room temperature. Distribution
of activity
in intracellular
CiS-AC-
“3”.
10 2a
Preparation
Glucose-grown Acetate-grown
onitate
Succinate counts/ counts/ min. min.
cells cells
1187 2185
40,440 ,91,&f
Fumarate
Malate
counts/ counts/ min.
min.
4,280 5,310
4,465 3,215
weight) containing
of
intermediates a-Ketoglutarate Citrate counts/ counts/ min. min.
61,440 661
11,650 4,320
a Experiments 1 and 2 were carried out at different times and are not to be compared with each other. Rather, the ratios of activities of succinate to a-ketoglutarate in each individual experiment are the significant observations in connection with the data presented in this table.
coli would bring about a condensation of two acetate molecules to succinate. No detectable succinate formed in the presence of either acetate alone or acetate and diphosphopyridine or triphosphopyridine nucleotide, adenosine triphosphate, coenzyme A or flavin adenine dinucleotide. Diphosphopyridine nucleotide enhances the reduction of methylene blue in the presence of acetate, but the reduction of the dye does not result in succinate formation from the added acetate. It, therefore, seems reasonable to conclude that E. wli does not contain an enzyme condensing acetate to succinate. Participation
of the Tricarboxylic
Acid Cycle in Glucose Oxidation
by
E. coli It was of interest to determine whether the tricarboxylic acid cycle participates in glucose oxidation by E. wli. Accordingly, a large quantity of resting cells was added to randomly labeled glucose and, at the end of incubation, the intracellular Krebs cycle intermediates were isolated and their activities determined. Both the di- and tricarboxylic acids became active, indicating their qualitative participation in glucose oxidation. Participation
of the Tricarboxylic Acid Cycle in Acetate Metabolism Growing Cells of E. coli
by
The participation of the tricarboxylic acid cycle intermediates in growing cells is evident from the data presented in Table V. Ten grams (wet weight) of cells was inoculated into 16 1. of a complete acetate
482
S.
J.
AJL
AND
D.
TABLE Participation
of the Tricarboxylic Growing Cells
Intermediate
Citrate a-Ketoglutarate Succinate Malate” Fumarate” cis-AconitateO (1 The
concentration
T.
of these
WONG
V
Acid Cycle of Escherichia
Concentration dJ
Could
0.
0.291 not be detected 143.3 intermediates
in Acetate coli
Metabolism
by
C” content counls/min.
Specific activity counlslmin. j&f
4,563 16,500 61,675 3,688 4,187 5,750
15,573
was not
430
determined.
medium to which were added 500 pM of acetate-2-Cl4 containing approximately 1 X 10’ counts/mm. After a 12-hr. period of incubation with constant aeration, 40 g. of cells was obtained. These were broken up, and the intracellular Krebs cycle intermediates were isolated and studied. It is quite evident from the data shown in Table V that all intermediates became radioactive, including cr-ketoglutarate.
DISCUSSION The experiments described in this, and in a recently published communication from our laboratories (12), coupled with those of Krampitz and co-workers (9, ll), leave no doubt that E. coli not only possesses all of the component enzymes of the tricarboxylic acid cycle but that it oxidizes acetate via this same mechanism. The complete equivalence between respiratory COZ and carboxyl carbons of both intracellular citrate and succinate during the oxidation of methyl-labeled acetate constitutes the major evidence for this assumption. The finding that a-ketoglutarate becomes highly active when radioactive acetic acid is oxidized by dried cells adds considerable weight to the hypothesis that this keto acid participates in the oxidization of the G-fatty acid. The failure to demonstrate a back-to-back condensation of two molecules of acetate to succinate in cell-free extracts of E. coli minimizes the possibility that a dicarboxylic acid cycle exists in this bacterium. It now appears that the earlier published data [recently summarized by Ajl (23)] dealing with (a) the equilibration of respiratory CO2 with succinyl carboxyl, (5) the failure of carrier citrate and cY-ketoglutarate to trap acetate carbon, and (c) quantitative data on 02 uptake and substrate disappearance during the oxidation of labeled acetate by nonprolifer.at-
TRICARBOXYLIC
ACID
CYCLE
IN
E.
COLI
483
ing cells were not sufficient to eliminate the tricarboxylic acid cycle as a terminal respiratory pathway in E. coli. On reinvestigating this problem by studying the intracellular intermediates of acetate oxidation (and not the externally added carriers), it becomes quite clear that citrate carboxyl, for example, not only equilibrates with respiratory COz but that the specific activity of the G-acid precurses succinate during the early stages of the experiment and falls below that of the Cd-acid toward the end of the experiment as the tricarboxylic acid cycle mandates. It should be noted that although carrier citrate as well as a-ketoglutarate becomes active during acetate-Cl4 oxidation by dried cells, these acids are not in equilibrium with respiratory COZ . The failure to demonstrate equivalance between respiratory COZ and carboxyl carbon of cY-ketoglutarate under any of our conditions is perplexing. Even when as many as 40 g. of cells was broken up, with the methodology employed by the authors, it was impossible to determine with any degree of accuracy any detectable intracellular a-ketoglutarate. The evidence for the participation of this keto acid in acetate oxidation comes, therefore, from the results of Swim and Krampitz (9) who succeeded in demonstrating equivalance between respiratory CO2 and intracellular a-ketoglutaric acid. Their findings, coupled with our observation that the band corresponding to the Cr,-keto acid on a paper chromatogram always contained some activity (making the specific activity of the acid extremely high and a potential precursor of succinate), does not justify the elimination of this keto acid as an intermediate in acetate oxidation by E. coli. The occurrence of the enzymes aconitase and isocitric dehydrogenase (N-12) adds considerable weight to this supposition, since these when combined convert quantitatively citric acid to c\l-ketoglutaric acid. The possibility exists that the enzyme which splits citric acid into Cd and Cz units, recently described by Ajl and co-workers (12, 24) and others (25, 26) or the enzymes which split cis-aconitate or isocitrate to succinate and glyoxylate recently found to be present in E. coli in our laboratories (27) and reported by Campbell et al. (28) to be present in Pseudomonas aemginosa (functioning with cis-aconitate) operates in this bacterium to such an extent that the bulk of tricarboxylic acid breakdown does not proceed via a-ketoglutarate but to succinate and acetate or glyoxylate directly. Although the tricarboxylic acid cycle has now been shown to occur in a number of microorganisms, it would be premature to conclude that it exists and operates in all of them.
484
S. J. AJL
AND
D. T.
0.
WONG
&JMMARY
By studying
the intracellular intermediates of acetate oxidation in equilibration between respiratory CO2 and carboxyl carbon of both citrate and succinate has been demonstrated. Dried preparations incorporate significant amounts of acetate carbon into all Krebs cycle intermediates. These data and those of Krampitz and co-workers, plus the finding that this organism contains a potent isocitric dehydrogenase and an aconitase, strongly suggest that E. wli does indeed respire via the tricarboxylic acid cycle. The failure to date to demonstrate an acetate-to-acetate condensing enzyme in cell-free extracts leaves much doubt whether this bacterium oxidizes acetate via an abridged type of cyclic mechanism, as the results with nonproliferating, resting cells previously suggested.
Escherichia coti, complete
REFERENCES 1. AJL, S. J., J. Bacterial. 69,499 (1950). 2. AJL, S. J., AND KAMEN, M. D., J. Biol. Chem. 189,845 (1951). 3. AJL, S. J., J. Gen. Physiol. 34,785 (1951). 4. AJL, S. J., KAMEN, M. D., RANSON, S. L., AND WONO, D. T. O., J. Biol. Chem. 189, 859 (1951). 5. AJL, S. J., AND WONO, D. T. O., J. Bacterial. 61,379 (1951). 6. SAZ, H. J., AND KRAMPITZ, L. O., J. Bacterial. 67, 409 (1954). 7. KAUFMAN, S., KOREES, S., AND DEL CAMPILLO, A., J. Biol. Chem. 192, 301 (1951). 8. STADTMAN, E. R., AND BARKER, H. A., J. Biol. Chem. 180,1169 (1949). 9. SWIM, H. E., AND KRAMPITZ, L. O., J. Bacterial. 67, 419 (1954). 10. BARBAN, S., “Enzymatic Mechanisms of Carbon Dioxide Assimilation in Bacteria.” Ph. D. dissertation, Washington University, 1953. 11. SWIM, H. E., AND KRAMPITZ, L. O., Federation Proc. 11,296 (1952). 12. WHEAT, R. W., AND AJL, S. J., Arch. Biochem. and Biophys. 49,7 (1954). 13. SWIM, H. E., AND KRAMPITZ, L. O., J. Bacterial. 67,426 (1954). 14. SANTER, M., AND AJL, S. J., J. Bacterial. 67,379 (1954). 15. GLOVER, J., KAMEN, M. D., AND VAN GENDEREN, H., Arch. Biochem. and Biophys. 36, 384 (1952). 16. MCILWAIN, H. J., J. Gen. Physiol. 2, ‘2% (1948). 17. KALNITSKY, G., UTTER, M. F., AND WERKMAN, C. H., J. Bacterial. 49, 595 (1945). 18. FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 19. SELIGSON, D., AND SHAPIRO, B., Anal. Chem. 24,754 (1952). 20. AJL, S. J., WONG, D. T. O., AND HERSEY, D. F., .7. Am. Chem. Sot. 74, 553 (1952). 21. NATELSON, S., PINCUS, J. B., AND LUGOVOY, J. K., f. BioE. Chem. 176, 745 (1948).
TRICARBOXYLIC
22. 23. 24. 26. 26. 27. 28.
ACID
CYCLE
IN
E.
COLI
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LARA, F. J. S., AND STOKES, J. L., J. Bacterial. 63, 415 (1952). AJL, S. J., Bacterial. Revs. 16, 211 (1951). WHEAT, R. W., WONG, D. T. O., AND AJL, S. J., J. Bacterial. 68, 19 (1954). GILLESPIE, D. C., AND GUNSALUS, I. C., Bacterial. Proc. 80 (1953). DAGLEY, S., AND DAWES, E. A., Nature 173, 345 (1953). WONG, D. T. O., AND AJL, S. J., to be published. CAMPBELL, J. J. R., SMITH, R. A., AND EAGLES, B. A., Biochim et. Biophys. Acta 11,594 (1953).