Regulation of metabolism in facultative bacteria

BIOCHIMICA ET BIOPHYSICA ACTA

33

BBA 25 496 R E G U L A T I O N OF METABOLISM IN FACULTATIVE BACTERIA II. EFFECTS OF AEROBIOSIS, A N A E R O B I O S I S AND N U T R I T I O N ON T H E FORMATION OF K R E B S CYCLE ENZYMES IN E S C H E R I C H I A

COLI

C. T. GRAY, J. W. T. WIMPENNY* AND M. R. MOSSMAN Department of Microbiology, Dartmouth Medical School, Hanover, N.H. (U.S.A.)

(Received March 2nd, 1965) (Revised manuscript received October 25th, 1965)

SUMMARY The levels of Krebs cycle enzymes were determined in Escherichia coli cells grown anaerobically and aerobically with varied nutrition. At least three factors were found to influence their biosynthesis: I, the presence or absence of molecular 02; 2, the repressive effect of glucose (catabollte repression) and 3, the balance between catabolic and anabolic demands on the cycle dictated b y the nutritional background. The lowest enzyme levels are found in anaerobically grown cells; however, both aerobic and anaerobic cells show further variations in enzyme content, depending upon the medium in which they are grown. The most dramatic nutritional effects are related to glucose metabolism. Glucose markedly represses the formation of Krebs cycle enzymes in cells grown on a complex medium, however, this repression is countermanded partially when cells are grown in a synthetic mineral salts medium with glucose in which the cycle must be used for synthetic purposes. When glutamate must be synthesized, the enzymes leading to its formation are elevated even in the presence of glucose; however, other enzymes of the cycle are not derepressed proportionately. I t is suggested that the cycle can be divided into at least three sectors which are under independent control. The data serve to emphasize that the enzymes of an amphibolic system, such as the Krebs cycle, i.e. one which is used for both catabolism and anabolism, are controlled b y a number of separate but interrelated mechanisms and that they cannot be regarded as so-called constitutive enzymes.

INTRODUCTION The Krebs cycle serves as a means for the oxidation of carbohydrates to CO 2 and H20. Early in its history it was recognized that the cycle could also serve synthetic functions, notably the synthesis of glutamate via ,,-ketoglutarate 1. While the cycle is associated primarily with aerobic life, certain portions of it are known to operate * Present address: Department of Microbiology, University College of South Wales and Monmouthshire, Cathays Park, Cardiff (Great Britain). Biochim. Biophys. Acta,

1I 7

(1966) 33-41

34

c . T . GRAY, J. w . T. WIMPENNY, M. R. MOSSMAN

anaerobically in this dual capacity. Thus facultative anaerobes growing anaerobically, e.g. Escherichia coli ~, or true anaerobes, e.g. Micrococcus lactilyticus 3, use the fourcarbon dicarboxylic acid portion of the cycle in reverse for anaerobic electron transport. Anaerobically grown cells may use the tricarboxylic acid portion of the cycle to form a-ketoglutarate and thence glutamate, when NH4+ is the sole source of nitrogen. The extensive use of the cycle by E. coli for synthetic purposes was deduced from the isotopic tracer studies of ROBERTS et al. 4. This subject was reviewed by WIAME 5, who discussed the possible anaerobic functions of the cycle. The Krebs cycle alone cannot account for the catabolic and anabolic use of 2-carbon compounds such as acetate and glycollate as a sole carbon source. To do so it must be supplemented by the glyoxylate cycle, in the case of acetate, and the dicarboxylic acid cycle and "glycerate pathway", in the case of glyoxylates. The principal function of the Krebs cycle under such circumstances varies according to the nature of the 2-carbon substrate. DAVIS7 has coined the term "amphibolic" to describe systems, such as the Krebs cycle, which are used for both anabolic and catabolic processes; he has also drawn attention to the complex and challenging problem of their regulation. It is obvious that the problem of understanding the control of an amphibolic system becomes even more complex when the nutrition and oxygen tensions are Varied. There is not much basic information available on this subject. Studies by ENGLESBERG et al.S, 9 using Pasteurella pestis, show that this facultative organism displays a greatly reduced activity toward Krebs cycle intermediates when grown anaerobically. A single complex medium, however, was used for both aerobic and anaerobic growth and the results do not instruct us on the question of amphibolic use of the cycle. UMBARGER1° did not examine as many enzymes as did ENGLESBERG and co-workers, but used synthetic and complex media in studying the changing levels of condensing enzyme accompanying the transition from anaerobiosis to aerobiosis in E. coli. COLLINS AND LASCELLES11 showed that the addition of glucose to nutrient broth produced Staphylococcus aureus cells which could not oxidize acetate, malate, and succinate. Extracts of such cells showed greatly reduced succinate and isocitrate dehydrogenase activity. Similar results were obtained by STRASTERS AND WINKLER1~ who showed that staphylococci grown on glucose broth oxidized Krebs cycle intermediates more slowly than when grown on plain broth. In the preceding paper of this series we compared the enzymes of aerobically and anaerobically grown E. coli with true aerobes and anaerobes from a standpoint of structure (location) and function 13. It was shown in passing that the Krebs cycle enzymes were decreased by anaerobic growth. It is the purpose of this paper to present extended data on these effects and to discuss the control mechanisms involved.

MATERIALS AND METHODS

Organism and media E. coli strain K12 was cultured on the synthetic or the complex (casein hydrolysate) medium described in the previous paper is. Sterile glucose, glycerol, gluconate, malate or pyruvate was added to either medium when desired, to give a concentration of 0.4 % (w/v). Biochim. Biophys. Aota,. Ix 7 (I966) 33-41

BIOSYNTHESIS

OF KREBS CYCLE ENZYMES IN

E. coli

35

Growth and preparation of cell fractions Cells were grown in io 1 of medium in large fermentors at 37 °, aerobically and anaerobically, under automatic pH control. The growth constant k was defined as the number of generations per hour. Complete details of methods used for growth, preparation of washed cells, cell disruption and separation of cell components into a cell-wall membrane and a soluble fraction have been described in the previous paper TM. Enzyme assays Condensing enzyme (citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7); aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3); isocitrate dehydrogenase (NADP+)] Ls-isocitrate: NADP oxido-reductase (decarboxylating), EC 1.1.1.42 ] ; succinate dehydrogenase (succinate: phenazine methosulphate oxidoreductase, EC 1.3.99.1); fumarase (L-malate hydro-lyase, EC 4.2.1.2); malate dehydrogenase (Lmalate: NAD + oxidoreductase, EC 1.1.1.37); and NADH oxidase were measured as described previouslyTM. The method of WILLIAMS AND HAGER 14, which measures pyruvate dehydrogenase manometrically by the liberation of CO 2 in the presence of ferricyanide, was used to determine a-ketoglutarate dehydrogenase. Glutamate dehydrogenase (Lglutamate: NADP + oxidoreductase (deaminating), ECI.4.I.4) was determined spectrophotometrlcally15. The oxidation of L-glutamate or a-ketoglutarate by whole cells was measured by conventional manometric technique using IO mM substrate in a 2.0 ml reaction system which was IOO mM with respect to phosphate buffer (pH 7.4) and 2.5 mM with respect to Mgz+. When manometry was used to assay enzymes in cells fractions, i.e. succinate dehydrogenase and a-ketoglutarate dehydrogenase, specific activities are expressed in ~1 of gas exchanged/mg protein/h at 3o °. The specific activities of all other enzymes were determined spectrophotometrically and are expressed as ~moles of substrate transformed/mg protein/h at 25 °. RESULTS AND DISCUSSION

Influence of oxygen on the level of Krebs cycle enzymes In the previous paper it was shown that the level of six Krebs cycle enzymes was lower in cells grown anaerobically on a salt-glucose medium than those grown aerobicallyTM. This is now shown to be the case when the simpler growth medium is replaced by a mixture of amino acids (complex) or when glucose is replaced by gluconate in the salt medium (Table I). Although it is clear that there is a general reduction in activity of Krebs cycle enzymes in anaerobically grown cells there is a wide range in variation between individual enzymes: c.f. aconitase and fumarase on the synthetic medium with glucose. These results are also supported by earlier work in which single enzymes have been studied, for instance, isocitrate dehydrogenase, aconitase, fumarase 9 and condensing enzym el°. The low but significant levels of Krebs cycle enzymes in anaerobically grown cells can be only partially explained. It is clear that in synthetically grown cells the Biochim. Biophys. Acta, xI 7 (I966) 33-41

~.~

.~

t~

t~

EFFECT

OF OXYGEN

ON THE

ACTIVITY OF KREBS

CYCLE ENZYMES

OF

E . eoli GROWN

ON

DIFFERENT

MEDIA

EFFECT

OF

NUTRITION

ON

KREBS

CYCLE

ENZYME

LEVELS

0.83 11.8 8.58 115 75.2 72 48.4

Growth k Condensing e n z y m e Aconitase Isocitrate d e h y d r o g e n a s e Succinate d e h y d r o g e n a s e Fumarase Malate d e h y d r o g e n a s e NADH oxidation

Soluble Soluble Soluble Cell-wall m e m b r a n e Soluble Soluble Cell-wall m e m b r a n e

Cell fraction

0.63 0.97 8"32 52 20.6 Ii 5.33

3 .o8 19 .1 85-3 231 50.8 225 38.2

4.9 19.7 1°-3 4.4 2.5 20. 4 7 .2 lO.4

o.73 3.5 8.5 54 30.0 17 8.8

4.6o 20.7 64.2 232 98. I 228 125

Aerobic

6.3 5.9 7.5 4.3 3-3 I3. 4 14.2 6.8

Ratio

Synthetic gluconate medium Anaerobic

fraction or the soluble fraction were determined by methods reported earlierTM.

3.5

7.7

3.3 8.6 2.1 1.2 1.6 4-5

Ratio

1.15 0.83 11.8 8.58 II 5 75.2 72 48.4

0.96 3-04 23. 9 55.9 I94 152 199 26.8

o.71 3.57 28.9 4°-3 284 128 I52 36.6

1.4I 9.20 41.8 32.8 226 142 I43 64.0

0.87 7.28 lO 4 70.7 518 237 I79 43.0

0.62 9.05 45.2 69.8 312 II3 371 40.8

0.87 3.o8 19.1 85.3 23i 50.8 225 38.2

0.42 5.7 ° 25.2 73.2 185 xoI 359 30.3

0.60 4.60 20. 7 64.2 232 98.I 228 I25

Complex Complex Complex Complex Complex Complex Synthetic Synthetic Synthetic glucose glycerol pyruvate gluconate malate glucose glycerol gluconate

Medium

Cells were disrupted and specific activities of the ce11-wall m e m b r a n e

THE

T A B L E II

Condensing e n z y m e 0.25 Aconitase 1.37 Isocitrate d e h y d r o g e n a s e 4.08 Succinate d e h y d r o g e n a s e 97 Fumarase 46.3 Malate d e h y d r o g e n a s e 16 N A D H oxidation 6.28 A v e r a g e ratio for all K r e b s ' cycle e n z y m e s

Aerobic

Synthetic glucose medium Anaerobic

Ratio

Anaerobic

Aerobic

Complex glucose medium

A c t i v i t y of K r e b s cycle e n z y m e s . R a t i o s of a n a e r o b i c to aerobic a c t i v i t y were c a l c u l a t e d b y t a k i n g t h e a n a e r o b i c a c t i v i t y of e a c h e n z y m e as u n i t y .

THE

TABLE I

>

~o

Cb

B I O S Y N T H E S I S OF K R E B S CYCLE E N Z Y M E S I N

E. coli

37

cycle must function to produce sufficient carbon skeletons for amino acid synthesis. Also in both synthetic and complex grown cells the dicarboxylic acid section of the cycle operates in the reductive pathway t o succinate TM. However, it is probable that all members of the cycle are synthesised to a significant extent whether or not they are employed by the cell. It is also of interest that NADH oxidase is reduced in anaerobic growth and not greatly affected by nutrition. Since these cells contain adequate amounts of cytochromes a 1, a~ and b~~a, it is likely that this reduction is due to a deficiency in some electron carrier other than these cytochromes.

The effect of glucose on the activity of Krebs cycle enzymes All the enzymes of the Krebs cycle tested were much reduced in aerobic cells grown on the complex media to which glucose was added (Table II). This is seen most clearly when the ratio of enzyme activity in the glucose containing medium is taken as unity and compared with that in the absence of glucose or when other substrates were added (Table III). The addition of glucose catabolites such as glycerol, pyruvate or gluconate did not reduce the enzyme activity as much as glucose. In contrast, the addition of a cycle intermediate such as malate increased the activity of Krebs cycle enzymes above that obtained on the complex medium alone (Table II). The effect of gluconate is of special interest in E. coli since in Aerobacter aerogenes the inhibitory effect of glucose is due to products of the gluconate pathway whereas these and previous experiments 17 show that gluconate is less effective in decreasing Krebs cycle enzyme activity than is glucose. The effect of glucose in decreasing the quantity of Krebs cycle enzymes is well documented. The early work of EPPS AND GALETMshowed that glucose greatly reduced succinate dehydrogenase activity. Later work by UMBARGER1°, COLLINS AND LASCELLES 11, HALPERN et al. TM and STRASTERS AND WINKLER TM added further evidence while MONODz0 studied the repression of the formation of inducible enzymes b y glucose as a model for the so-called "glucose effect". NEIDHARDTAND MAGASANIKzl and later MAGASANIK~ proposed that the pool of catabolites formed from glucose repressed the formation of enzymes concerned with their formation and MAGASANIK~ proposed the name "catabolite repression" as a general one for this effect. This term has an advantage in that it recognizes that compounds other than glucose or related hexoses can lead to repression~lm -25. The question that the present results raises sharply concerns the mechanism of repression by glucose catabolites of enzymes which themselves diminish the catabolite pooh "Catabolite repression" and amphibolic use of the cycle The role of the Krebs cycle in synthesis would be expected to be fully expressed during growth on the synthetic salt mixture with either glucose or one of its catabolites as carbon source and NH4 + as the sole nitrogen source. Under such conditions UMBARGERTM found that even in the presence of glucose, enzymes such as the condensing enzyme, aconitase and isocitrate dehydrogenase concerned with glutamate synthesis reached higher levels of activity than in the complex medium. This is consistent with the finding that the tricarboxylic acid/dicarboxylic acid ratio increased (Table II) under such conditions. It has also been found that another enzyme concerned in amino acid synthesis, namely glutamate dehydrogenase, also increases Biochim. Biophys. Acta, 117 (I966) 33-41

I

~o O~

h~

'

~"

EFFECT

OF NUTRITION

ON

KREBS

CYCLE ENZYME

LEVELS

EFI~ECT OF GROWTH

CONDITIONS

ON

0.96 3.7 2.0 6. 5 1. 7 2.0 2.8 3.1 1.9 0.55

SYSTEMS METABOLIZING

1.15 i.o I.O I.O 1.o i.o I.O I.O I.o I.O

Complex glycerol

GLUTAMATE

o.71 4.3 2. 4 4.7 2. 5 1. 7 2.1 2.9 1.8 0.76

Complex pyruvate

AND

1.41 Ii.i 3.5 3 .8 2.0 1. 9 2.o 4.0 3.1 1.3

o.87 8.8 8.7 8.2 4-5 3.1 2. 5 6.0 2. 5 o.89

Complex malate

~-KETOGLUTARATE

Complex gluconate o.62 ii 3 .8 8.1 2.7 1. 5 5.1 5.4 2.5 0.84

Complex

o.87 3.7 1.6 IO 2.0 0. 7 3.1 3 .6 2.7 o.78

Synthetic glucose

o.42 6.9 2.1 8.5 1.6 1. 4 4.9 4 .2 2.2 o.63

Synthetic glycerol

o.6o 5.5 1. 7 7.5 2.o 1. 3 3.2 3.5 2.3 2.6

Synthetic gluconate

Glutamate oxidation Glutamate dehydrogenase ~-Ketoglutarate oxidation ~-Ketoglutarate dehydrogenase

0. 7 18.8 0. 4 o 0. 4 1.47 o 5.6

3.1 1.27 14 22

Complex glucose

Synthetic glucose

Complex glucose

Aerobic

Anaerobic

1. 3 1.2o 23 79

Complex

2.2 19.6 17 23

Synthetic glucose / * 1 0 z / m g d r y w t. p e r h /~moles/mg p r o t e i n p e r h # 1 0 J m g d r y wt. p e r h /~1 C O J m g p r o t e i n p e r h

Units

O x i d a t i o n of s u b s t r a t e s w as d e t e r m i n e d m a n o m e t r i c a l l y i n w h o l e cells; d e h y d r o g e n a s e a c t i v i t y w a s m e a s u r e d i n cell free e x t r a c t s ; specific a c t i v i t i e s were d e t e r m i n e d as described in METHODS.

THE

T A B L E IV

Growth k C on den s in g e n z y m e Aconitase Isocitrate dehydrogenase Succinate dehydrogenase Fumarase Malate dehydrogenase A v e r a g e r a t i o for al l K r e b s cycle e n z y m e s Tricarboxylic acid/dicarboxylic acid ratio NADH oxidation

Complex glucose

Growth condition (aerobic)

Cells were d i s r u p t e d a n d specific a c t i v i t i e s of t h e c e l l - w a l l m e m b r a n e f r a c t i o n or t h e s o l u b l e f r a c t i o n w e r e d e t e r m i n e d b y m e t h o d s r e p o r t e d e a r l i e r 13. All r a t i o s were d e t e r m i n e d from t h e d a t a b y s e t t i n g t h e v a l u e for e x t r a c t s of E. coli g r o w n a e r o b i c a l l y on t h e c o m p l e x m e d i u m w i t h g lu co s e a t i.o. The t r i c a r b o x y l i c a c i d / d i c a r b o x y l i c a c i d r a t i o s e x p r e s s t h e a v e r a g e l e v e l s of e n z y m e s a t t a c k i n g t r i c a r b o x y l i c a c i d s t o t h o s e for e n z y m e s a t t a c k i n g d i c a r b o x y l i c acids, a n d are dis c us s ed i n t h e t e x t .

THE

TABLE III

~nO

t~

t~o

BIOSYNTHESIS

OF

KREBS

CYCLE

ENZYMES

IN

E. coli

39

(Table IV) on the salt medium: this enzyme works more rapidly in the reverse direction, i.e. as glutamate synthetase. These results are best interpreted as an alleviation of catabolite repression, i.e. derepression b y a synthetic demand. NEIDHARDT AND MAGASANIK2e found a similar situation in studies on histidine catabolism. One other characteristic of catabolite repression which is of interest is the relationship between it and growth rate where it has been found that repression varies inversely with growth rate23, 27. In the present work no such consistent relationship between growth rate and repression b y glucose has been found (Table V). All nutrient conditions yielding growth rates lower than that on the complex medium with glucose, however, had higher levels of Krebs cycle enzymes. TABLE

V

R E L A T I O N S H I P B E T W E E N GROWTH RATES AND K R E B S CYCLE E N Z Y M E FORMATION IN A E RO B IC AL L Y ON VARIOUS M E D I A

E. coli GROWN

Medium

Growth k

d verage ratio A verage ratio for all enzymes for tricarboxylic acid enzymes

Average ratio for dicarboxylic acid enzymes

Tricarboxylic acid] dicarboxylic acid

Complex gluconate Complex glucose Complex glycerol Synthetic glucose Complex malate Complex pyruvate Complex Synthetic gluconate Synthetic glycerol

1.41 1.15 0.96 o.87 o.87 o.71 0.62 0.6o 0.42

4.0 i.o 3.1 3.6 6.o 2.9 5.4 3-5 4.2

2.0 I.o 2.2 1.9 3-4 2.1 3-I 2.2 2.6

3.1 I.o 1.9 2.7 2.5 1.8 2.5 2.3 2.2

6.1 i.o 4.1 5.1 8.6 3.8 7 .6 4.9 5.8

I t seems reasonable to explain these results b y assuming that with adequate glucose, in an organism such as E . coli with a high aerobic and anaerobic rate of glycolysis, enough energy (ATP) is available from the Embden-Meyerhof pathway to minimise the role of the Krebs cycle in producing energy. Under these conditions the synthetic role of the cycle predominates. This too will be of lesser importance when amino acids derived from the cycle are present in the medium so that the full repressive effect of glucose will be seen on the complex medium. With a carbon source such as malate the cycle will be used mainly for energy production and this will explain the high activities of Krebs cycle enzymes found with this substrate. The data is also consistent with the idea that the cycle enzymes are induced or repressed in three main groups, namely the enzymes metabolising: a, the tricarboxylic acids; b, the 5-carbon dicarboxylic acids; c, 4-carbon dicarboxylic acids. All m a y be considered as being induced or derepressed on the complex medium without glucose, under conditions in which energy is produced b y the cycle and there is no need to synthesize the glutamate or aspartate family of amino acids: glutamate dehydrogenase activity thus remains low, i.e., is repressed. On the synthetic medium with glucose and N H , + as nitrogen source Groups a and c are derepressed whereas a-oxoglutarate dehydrogenase in Group b remains repressed because its substrate is required for synthetic purposes. Biochim. Biophys. Acta, Ix 7 (1966) 33-41

40

C . T . GRAY, J. W. T. WiMPENNY, M. R. MOSSMAN

Under anaerobic conditions energy is produced by glycolysis and low but significant levels of Krebs cycle enzymes are still found. Those of the tricarboxylic acid cycle in Group a are employed on a synthetic medium for the synthesis of 5-carbon skeletons whereas in both synthetic and complex media enzymes of Group c can function in the reductive dicarboxylic acid pathway to succinate. There is growing evidence that the reductive formation of dicarboxylic acids constitutes a biosynthetic pathway and an electron acceptor system involving the use of alternate forms of enzymeslS,28,zL The enzymes of Group a may be under further individual control mechanisms as is indicated by the large increase in activity of condensing enzyme and others when cells are grown on z-carbon compounds such as acetatee, 3°. It is likely that feed-back inhibition mechanisms would also play a role in vivo in the balance of these reactions. These proposed control mechanisms together with detailed genetic analysis seem well worthy of further examination. It is clear, however, that there is now ample experimental support for the growing agreement that not only are the differences between catabolic and anabolic enzymes largely artificial 31, but that this is also true of the differences between so called "inducible" and constitutive enzymes. ACKNOWLEDGEMENTS

The authors express their gratitude to Professor D. E. HUGHES for his help and advice during the preparation of this manuscript, also to Mr. A. T. E. MORRISSEY for technical assistance. This investigation was supported by a grant (AI-o5 806) from the Institute of Allergy and Infectious Diseases of the National Institutes of Health, U.S. Public Health Service. One author (J. W.T. W I M P E N N Y ) w a s partially supported by training grant 5T1-GM 961 from the National Institutes of Health.

REFERENCES I 2 3 4 5 6 7 8 9 1o 11 12 13 14 15 I6 17 I8 19 20 21

KREBS, S. GURIN AND L. V. EGGLESTON, Biochem. J., 5 x (1952) 614. KREBS, Biochem. J., 31 (1937) 2095. P~CK, JR., O. H. SMITH AND H. GEST, Biochim. Biophys. Acta, 25 (1957) 142. ROBERTS, P. H. ABELSON, D. B. COWlE, E. T. BOLTON AND R. J. BRITTEN, Carnegie Inst. Wash. Publ., 6o 7 (1955) 218. J. M. WIAME, in F. F. NORD, Advan. Emymol., Vol. I8, Interscience, N e w York, x957, p. 241. H. L. KORNBERG, Cold spring Harbor Syrup. Quant. Biol., 26 (I961) 257. B. D. DAvis, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) I. E. ENGLESBEEG, A. GIBOR AND J. B. LEVY, J. Bacteriol., 68 (I954) 146. E. ENGLESBERG, J. B. LEVY AND A. GIBOR, J. Bacteriol., 68 (1954) 178. H. E. UMBAEGER, J. Bavteriol., 68 (1954) 14o. F. M. COLLINS AND J. LASCELLES, J. Gen. Microbiol., 29 (1962) 53I. K. C. STRASTERS AND K. C. WINI~LER, J. Gen. Microbiol., 33 (1963) 213. C . T . GRAY, J. W . T. WIMPENNY, D. E. HUGHES AND M. R. MOSSMAN, Biochim. Biophys. Acta, I17 (I966) 22. F. R. WILLIAMS AND L. P. HAGER, Biochim. Biophys. Acta, 38 (196o) 566. W . S. HALPEEN AND H. E. UMBARGER, J. Bacteriol., 80 (196o) 285. C. A. HIRSCH, M. RASMINSKY, ][3. D. DAVIS AND E. C. C. LIN, J. Biol. Chem., 238 (1963) 377 o. M. COHEN AND K. HORIEATA, J. Bacteriol., 78 (1959) 624. H. ~¢~, R. EPPS AND E. F. GALE, Biochem. J., 36 (1942) 619. Y. S. HALPERN, A. EVEN-SHOSHAN AND M. ALTMAN, B~o'rhim. Biophys. Acta, 93 (1964) 228. J. MONOD, Recherches sur la Croissance des Cul{ures Bacteriennes, H e r m a n n , Paris, 1941. F. C. NEIDHARDT AND B. MAGASANIK, Nature, 178 (1956) 8oi. H. H. H. R.

A. A. D. B.

Biochim. Biophys. Aaa, xr 7 (1966) 33-41

BIOSYNTHESIS OF KREBS CYCLE ENZYMES IN E. coli

41

B. MAGASANIK, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) 249. J. MANDELSTAM, Biochem. J., 82 (1962) 489. E. MCFALL AND J. MANDELSTAM, Nature, 197 (1963) 88o. K. PAIGEN, Biochim. Biophys. Acta, 77 (1963) 318. F. C. lX]'EIDHARDT AND B. MAGASANIK, J. Bacteriol., 78 (1957) 253. E. R. TUSTANOFF AND W . BARTLEY, Biochem. J., 91 (1964) 595. F. PICHINOTY AND G. COUDERT, Experientia, 18 (1962) 257. C. 1R. AMARASINGHAM, Federation Proc., 23 (1964) 487. H. L. KORNBERG AND S. R. ELSDEN, in F. F. NORD, Advan. Enzymol., Vol. 23, Interscience, N e w York, 1961, p. 4Ol. 31 H . E. UMBARGER, Ann. Rev. Plant Physiol., 14 (1963) 19.

22 23 24 25 26 27 28 29 30

Biochim. Biophys. Acta, 117 (1966) 33-41