Effect of glucose on the utilization of succinate and the activity of tricarboxylic acid-cycle enzymes in Escherichia coli

228

BIOCHIMICA ET BIOPIIYSICA A{;TA

BBA 25136

E F F E C T O F G L U C O S E ON T H E U T I L I Z A T I O N O F S U C C I N A T E A N D T H E A C T I V I T Y OF T R I C A R B O X Y L I C A C I D - C Y C L E E N Z Y M E S IN E S C H E R I C H I A

COLI

Y E H E S K E L S. H A L P E R N , A V I V I T H E V E N - S H O S H A N AND M I C H A E l . ARTMAN

Departme~,t of BaclerioloKv, Hebrew Universih'-Hadassah Medical School, .Jerusalem (Israel) (Received March 6th, 1964)

SUMMARY

When cultures of Escherichia coli growing exponentially in a glucose--salts medium were transferred to a similar medium containing succinate as the sole source of carbon, a lag in growth of about 4 h was observed. The rate of O,, uptake by non-proliferating suspensions of glucose-grown cells in the presence of succinate was about 9 ?o of that observed with succinate-grown cells. The rate of O z uptake by cell-free extracts of glucose-grown cells in the presence of succinate (and other tricarboxylic acid cycle intermediates) and the activity of tricarboxvlic acid-cycle enzymes in these extracts were 2-4-fold lower than the rate of O e uptake and the corresponding enzymatic activities in extracts of succinate-grown cells. The intracellular concentration of "succinate" following incubation with [~4Csuccinate was 3-5-fold higher in succinategrown than in glucose-grown cells. It is concluded that the lag in growth observed upon transfer of glucose-grown E. coli to a succinate medium is a combined effect of the repression byglucose of tricarboxylic acid-cycle enzymes and the active transport of succinate into the cell.

IN'IROI} U(?TION

ROBERTS et al), have shown that Escherichia coli grown in a synthetic medium in which glucose served as the sole source of carl)on did not resume growth at once when transferred to media containing a variety of substrates as the principal source of carbon. The longest lag was obtained when the cells were transferred to a suecinate medium. The authors showed, however, that when E. coli grew on glucose about 3o% of the total amount of CO z evolved by these organisms came from the Krebs cycle. That glucose-grown E. coli do not readily metabolize externally added succinate can be inferred from a recent study on the effect of succinate on the induced synthesis of ~8-galaetosidase (/3-D-galactoside galactohydrolase EC 3.2.2.23) by glucose-grown and succinate-grown cells 2. The results obtained in this study showed that, in agreement with the finding of MAXDELS'rAMa, succinate repressed the formation of/3-galactosidase by non-proliferating succinate-grown cells. On the other band, succinate was without any effect on the induced synthesis of B-galactosidase by non-proliferating glucosegrown cells. Hiochim. Biophys..4cta, 93 (1964) _,28-_,36

SUCCINATE UTILIZATION BY GLUCOSE-GROWN E. coli

229

It seemed, therefore, of some importance to studv the utilization of succinate by glucose-grown cells and to attempt to elucidate the reason for the lag in growth of glucose-grown cells upon transfer into media with succinate as the sole source of carbon. MATERIALS AND METHODS

Organisms and media. E. coil strain H (Harvard) was used throughout. The bacteria were grown in the synthetic medium of DAVIS AND MINGIOLI4 supplemented with 0.5 % glucose or succinate, as indicated. Growth took place in a Dubnoff water bath at 37 ° and a shaking rate of IOO oscillations per rain. Growth curves. To prepare growth curves the bacteria were grown in ioo-ml flasks provided with side arms for direct measurement of absorbancy. Viable counts were made by plating out I ml of a suitable cell dilution on Difco nutrient agar plates. Dry weight of cells was determined by weighing suspensions heated at I IO° to constant weight. A standard curve relating density to viable count and dry weight was prepared. Preparation of non-proliferating suspensions. Cells from the exponential phase of growth were collected by centrifugation and washed twice with a o.85 % NaC1 solution. The washed cells were resuspended in fresh medium to the desired absorhancv as measured in a Coleman Jr. spectrophotometer at 55o m/*. Preparation of cell-free extracts. The organisms were grown as described above. After harvesting and washing, the cell suspension was subjected to sonic vibrations in a IO KC Raytheon Magnetostriction Oscillator for 3 min and centrifuged at 18 ooo × g for IO min in a refrigerated RC-2 Sorvall centrifuge to sediment unbroken cells and cell debris. The cell-free supernatant was used. The protein content of cell-free extracts was determined bv the biuret method of MEHI.5 using a Coleman .Jr. spectrophotometer at 54o m/*. Manometric determinations. Oxygen consumption of non-proliferating suspensions and cell-free extracts was measured by the conventional Warburg technique at 37 ° in an air atmosphere. In experiments on the utilization of [~*C!succinate the centre wells of the Warburg vessels contained a 15 % solution of CO2-free KOH and the expired ~*CO~was trapped directly into this solution. The amount of CO2 released was calculated from the amount of O 2 taken up. At the end of the experiment, the contents of the centre wells were quantitatively transferred to stoppered centrifuge tubes to which IO mg of carbonate carrier were added. The carbonate was precipitated by dropwise addition, with stirring, of an excess of 12 % BaC12. The barium carbonate formed was sedimented by centrifugation and the sediment washed several times to remove all traces of alkali. The washed BaCO a precipitate was dried in vacuo and the dry powder mounted on o.3-cm 2 disks and counted at infinite thickness with a thin end-window Geiger-Miiller tube. Specific radioactivities were obtained by direct comparison with a poly-[14C]methacrylate standard (I/,C/g) obtained from the Radiochemical Centre, Amersham, Bucks. The standard error in all the determinations did not exceed 5 %- [14CiSuccinate was obtained from the Radiochemical (;entre, Amersham, Bucks. Assay of tricarboxylic acid-cycle enzymes Acetate kinase (EC 2.7.2.1 ). The activity of acetate kinase was determi'ned according to ROSE et al3. The reagent of LIPMANN AND TIITTLE7 was used in order to Biochim. Biophys. Acta, 93 (I964) 228-236

230

v.s.

tlALPERN,

A. E V E N - S H O S H A N ,

M. A R T M A N

o b t a i n the coloured fe.rric h y d r o x a m a t e complex. One unit of e n z y m e is t h a t a m o u n t of e n z y m e which produces I / , m o l e of h y d r o x a m i c acid per min. Aconitate h3,dratase (EC 4.2.I.3). A c o n i t a t e h y d r a t a s e was d e t e r m i n e d b y the m e t h o d of R.~CKER~. One unit is defined as t h a t a m o u n t of e n z y m e which causes an initial rate of increase in a b s o r b a n c y (at 24o m/~) of o.ooI per min. Isocitrate dehydrogenase (EC r.r.I.41 ). I s o c i t r a t e d e h y d r o g e n a s e was d e t e r m i n e d bv the m e t h o d of KORNBER(; AND PRICER 9. One unit of e n z y m e is t h a t a m o u n t of e n z v m e which causes the reduction of x #.mole of N A D P in 2o rain. Succinate dehydrogenase (E(" 1.3.99.I). Succinate d e h y d r o g e n a s e was d e t e r m i n e d b y the phenazine m e t h o s u l f a t e m e t h o d of BI,.'RNATIt ..~.XD SINGER 1°. One unit of e n z y m e is defined as t h a t a m o u n t of e n z y m e which causes the u p t a k e of x p,l of O~ per rain. Irumarate hydratase (EC 4.2.i.2). F u m a r a t e h v d r a t a s e was d e t e r m i n e d according to R.~CKF.10L One unit is t h a t a m o u n t of e n z y m e which causes an initial rate of change in a b s o r b a n c y (at 24o mt,) of o.oI per min. Malate dehydrogenase (EC 1.1.1.37 ). E n z v m e a c t i v i t y was d e t e r m i n e d according to MEHI.ER el al. 12. One unit is defined as t h a t a m o u n t of e n z v m e which causes a decrease in a b s o r b a n c v of o.oI per min. All e n z y m e activities are expressed as units per mg protein.

Determination of intracelhdar succinate. Cultures growing e x p o n e n t i a l l y on glucose a n d on succinate as the respective sole source of carbon were h a r v e s t e d by c e n t r i f u g a t i o n a n d r e s u s p e n d e d to a d e n s i t y of o.45 in fresh m e d i u m from which the carbon source was o m i t t e d . 2-ml s a m p l e s were i n c u b a t e d at 37 ~ w i t h o u t shaking. A f t e r 3 rain o.2 ml of a c h l o r a m p h e n i c o l solution (I mg/ml) was a d d e d and incubation c o n t i n u e d for a d d i t i o n a l 2 min. V a r y i n g a m o u n t s of ~14Qsuccinate (specific a c t i v i t y - o.o087/xC/tLmole) were a d d e d to the incubation m i x t u r e s and the tubes were i n c u b a t e d for a d d i t i o n a l 4 ° rain. The time of i n c u b a t i o n was chosen on the basis of p r e l i m i n a r y e x p e r i m e n t s which showed t h a t the u p t a k e of label occurred very r a p i d l y a n d the intracelhflar r a d i o a c t i v i t y r e m a i n e d p r a c t i c a l l y c o n s t a n t t h r o u g h o u t the entire period e x a m i n e d (2-4o min). At the end of i n c u b a t i o n the cells were washed b y centrifuging until no a d d i t i o n a l counts were released by the cells. F o r this purpose 4 washings with 2-ml p o r t i o n s of basal m e d i u m were sufficient. The washed bacterial pellet was resusp e n d e d in distilled w a t e r a n d the suspension boiled for 2o min. The cells were r e m o v e d by eentrifugation a n d the s u p e r n a t a n t d e c a n t e d into a l u m i n u m lflanchets, e v a p o r a t e d to drvness a n d r a d i o a c t i v i t y m e a s u r e d with a Nuclear-Chicago gas-flow thin-window ctmnter w i t h o u t corre, ction for self-absorption. Intracelhflar c o n c e n t r a t i o n s of "succin a t e " are expressed a s / , m o l e s of suceinate per ml of intracelhflar water, as calculated from m e a s u r e m e n t s of r a d i o a c t i v i t y a n d e x p e r i m e n t s relating a b s o r b a n c v of bacterial suspensions to d r y a n d wet weight of cells. I ml of A.sa0 rnt~ o.70 was e q u i v a l e n t to x mg d r y weight or o.oo 3 ml of intracelhflar water. R E S U I.TS

Growth experiments (;lucose-grown cells were inoculated into m e d i a c o n t a i n i n g succinate as the sole source of carbon a n d g r o w t h was followed as described in MATERIALS AND METHODS. At different p o i n t s on the g r o w t h curve s a m p l e s were t a k e n to d e t e r m i n e the rate of respiration in the presence of succinate. A t y p i c a l e x p e r i m e n t is i l l u s t r a t e d in Fig. r.

Biochim. t]tophys. Acta, 03 (r964) --8 23,,

SUCCINATE IJTILIZATION BY GLUCOSF-GROWN

E. coli

23I

One can see that there was a lag of about 4 h before the bacteria began to grow in the new medium. The rate of oxygen uptake in the presence of succinate, which was very low at the beginning, increased gradually during the lag period, reaching a steady-state level when the culture entered the logarithmic phase of growth. On the other hand, cells transferred into a succinate medium from acetate resumed logarithmic growth after a short lag of about 15 min.

!

/ '

,/

o,

,.oIA8

,4

~08

, ol

/

0.06 0.041 0

|.1

60 , 1

, 2

"Iv 3

0~ 4 5 HOURS

6

7

I

2 3 4 SAMPLE

5 No

6

Fig. I. 1"he r a t e of g r o w t h of E. cell on s u c c i n a t e following t r a n s f e r from glucose a n d a c e t a t e m e d i a C u l t u r e s of 1.2. cell g r o w i n g e x p o n e n t i a l l y on glucose a n d on a c e t a t e as t he sole source of c a r b o n were h a r v e s t e d , w a s h e d a n d t r a n s f e r r e d to a s u c c i n a t e m e d i u m a n d i n c u b a t e d w i t h s h a k i n g a t 37:. G r o w t h was followed by t u r b i d i t y m e a s u r e m e n t s (- × --, glucose-grown i n o c u l u m ; - - t , acetateg r o w n i n o c u l u m ) . At p o i n t s on the g r o w t h c u r v e i n d i c a t e d by a r r o w s (the n u m b e r s in circles d e s i g n a t e t h e c o n s e c u t i v e samples) s a m p l e s were w i t h d r a w n , w a s he d a n d used for m e a s u r i n g o x y g e n u p t a k e : w i t h no s u b s t r a t e a d d e d (open bars); in t he pre s e nc e of o.o- M glucose (da s he d b a r s ) : in the presence of o.o2 31 s u c c i n a t e (solid bars). For o t h e r e x p e r i m e n t a l c o n d i t i o n s , see M A T E R I A L S A N D M E T H O D S a n d T a b l e I.

Utilization of [t4Cqsuccinate The very low capacity of non-proliferating glucose-grown E. coli for respiration on succinate has been clearly demonstrated in experiments with [14C':succinate. The results of these studies are presented in Table I. These data show that the rate of oxygen uptake by succinate-grown cells in the presence of succinate was more than IO times as high as the rate of endogenous respiration. The good agreement between the amount of ~4CO2 found and the CO.2 released as calculated from O 2 uptake, indiTABI.E I U T I L I Z A T I O N OF [ I l C ' s u C C I N A T E

BY NON-PROLIFERATING

SUSPENSIONS

()F 1£. c e l l

G R O W N ON G L U C O S E A N D S U C C I N A T E AS ] ' H E S O L E S O U R C E O F C A R B O N

E a c h flask c o n t a i n e d : 0. 5 a b s o r b a n c v u n i t of b a c t e r i a l s u s p e n s i o n , i.o ml ; o.I M p h o s p h a t e buffer (pH 7.2), 0.7 m l ; [5°~, K O H in t h e c e n t r e well, o . , ml ; o.I 5 M u n i f o r m l y labelled q'W s u c c i n a t e ( o. oz/tC//tmo le), o. 3 ml t i p p e d from t h e side a r m a f t e r e q u i l i b r a t i o n ; final vol ume , 2.2 ml (ma de up w i t h d i s t i l l e d water). F o r o t h e r e x p e r i m e n t a l c o n d i t i o n s , see MATERIALS AND METHODS.

Experiment :",'o.

i 2 3 4

N¢,n-prohferating suspenst~}ns of cells grown on

Glucose Glucose Succinate Succinate

Oxygen uptake (lonoles/.lo rain) Endogenous

o.35 o.27 o.42 0.33

With succinatc

0.37 o.36 4.2o 4.18

COt release (t, moh.s] ~,) rain) Calculated ]'rora tff'O t oxygen uptake found

o.-12 o.4 t 4.9o 4.7 °

o.38 0.42 6.o0 4.9o

Biochim. Btophys. Acla, 93 (t964) 2 2 8 - z 3 6

232

Y.S.

HALPERN,

A. E V E N - S H O S H ' A N ,

M. A R T M A N

cates that the oxidation of succinate proceeded to completion. On the other hand, with glucose-grown cells the rate of succinate oxidation was very low, only slightly above the endogenous, amounting to about 9 O/,,, of that observed with cells grown on succinate.

Oxygen uptake in the presence of different substrates Tal)le II presents the results of experiments in which the rate of oxygen uptake by non-proliferating suspensions of glucose-grown and succinate-grown E. coli was examined in the presence of different carbon compounds. Both suspensions exhibited similar rates of O z uptake in the I)resence of glucose. In the presence of lactate and pyruvate the rate of oxygen uptake by succinate-grown bacteria was 1.5 -2-fold higher "F.\ H I . E 11 RESPIRATION

OF NON-PROLIFERATING

E.

SUSPENSIONS

coli IN THE PRESENCE

OF SUCCINATE-

OF VARIOUS

AND GLUCOSE-GRO~,VN

SUBSTRATES

E a c h f l a s k c o n t a i n e d , in a d d i t i o n t o n o n - p r o l i f e r a t i n g s u s p e n s i o n s a n d p h o s p h a t e b u f f e r (see T a b l e I), 6 o t t m o l e s of t h e s u b s t r a t e i n d i c a t e d (or 12o p m o l e s of t h e r a e e m a t e ) . F o r o t h e r e x p e r i m e n t a l c o n d i t i o n s , see T a b l e I. 0.~ vecn uptake (pl Ot per .4 Experiment So.

I 2

3 4 5

Endogtnous

3 3 2 3 .!

o.z per r 5 m i n i in the presence of:

Glucose Nt¢cctnah" DL- 3lalate 2,.O.t'oglularate Pyruvate DL. Lactate . . . . . . . . . . . . . . . . . . . . . . . . . . . glucose succinate gilt/dose SlI.CCi?IaIt" glucose succlnale glucose succinate glucose succinate glucose succmat, grown grown grown grown grcaz*t groum grottm grou'n grown grown grme, n gr~u.n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 10 IO 2t t4

It) 17 ~7 2t 14

3 7 5 3 .I

37 30 48 -t 2 3s

5 4 4 5 13

"o 21 3t 30 2~

S 4 2 7 3

7 O 5 ~ 4

12 15 2I 18 13

2() 31 29 3° -'7

19 I(l 15 tS I-'

33 3I 3' 3" 31

than that found with glucose-grown cells. On 2-oxoglutarate as the substrate both suspensions displayed similar, low rates of 0 2 uptake (only twice as high as the endogenous rate). However, with succinate and malate as substrates there was a striking difference in the behavior of glucose-grown and succinate-grown cells. The oxygen ut)take by glucose-grown ce.lls in the presence of these substrates was only slightly above the values of endogenous respiration. With succinate-grown cells the rate of 0,, uptake in the presence of malate was approx. 5-fold higher, and in the presence ~,f succinate 9-fold higher than with gltmose-grown (:ells.

Oxygen uptake by cell-free extracts In order to test the possibility that the difference between glucose-grown and succinate-grown cells is one of permeability to the substrates tested, we examined the oxygen uptake by cell-free extracts of the two cultures in the presence of different substrates. When cell-free extracts were used, the differences in the rates of oxygen uptake by preparations from glucose-grown and succinate-grown cells in the presence of malate and succinate were considerably smaller than with whole cell suspensions (1:2.-3 in extracts versus 1 : 5 - 9 in whole cells). In the presence of lactate and pyruvate the rate of O z uptake by extracts from succinate-grown cells was 2- 3 times as high as with extracts from glucose-grown cells. These differences in activity between the two Btochim. Biophys. Acta, 9 3 0 9 6 - I ) , , S 2 3 ~

SUCCINATE UTIIAZATION BY GLUCOSE-GROWN E. coli

233

extracts were similar to those found with whole cell suspensions. A marked difference between whole cell suspensions a n d extracts in response to 2-oxoglutarate was noted. As shown in Table I I the rate of O 2 u p t a k e b y non-proliferating suspensions of both glucose- a n d suecinate-grown cultures in the presence of 2-oxoglutarate was very low, only slightly above t h a t of endogenous respiration. On the other hand, with cell-free extracts the rates of oxygen u p t a k e in the presence of 2-oxoglutarate were similar to those o b t a i n e d with p y r u v a t e . As with p y r u v a t e , extracts from succinate-grown cells exhibited a 2 -3-fold higher rate of 02 u p t a k e t h a n extracts from glucose-grown cells. \Vith glucose as s u b s t r a t e the rate of Oz u p t a k e b y cell-free extracts from succinategrown cells was even slightly below t h a t found with extracts from glucose-grown cells. I t should be m e n t i o n e d t h a t a 2-fold dilution of the extracts (from 5 to 2. 5 mg protein per reaction mixture) resulted in complete a b o l i s h m e n t of respiration in the presence of glucose. TABI.E IIl RESPIRATION

OF CELL-FREE

E X T R A C T S OF S U C C I N A T E -

]".'. colt' I N T H E P R E S E N C E

AND GI.UCOSE-GROXVN

OF VARIOUS SUBSTRATES

All values have been corrected for endogenous respiration. For experimental conditions, see Table II. Ox)'~cn uptake (lit 0 i per 70 rain) in the presence of: F. ttract protein per tessel, mg

.guccinate Ghu=ose DL-Malatc . . . . . . . . . . . g/l~cos¢ succinate glucose succinale glucose succinat¢ gr¢~,~ groum grown grown gr~vn gro~

5.00 2.5o L25 o.6o 0.30 o. x 5

lo 7 87 47 3° I3 6

252 2x 7 Ill

76 4° i z

88 o

65 0

2-Oxoglutaratc DL-LactaIc . . . . . . . glucose" succinate glucose succinate grown gro~ grow~ grou~

29 8~ 22 13 43 Io o o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

l)vruvat¢ glucose succinat, grow~ gr~,n

49 ~43 283 25 3° 49 I43 -. . . . . . . . . . . . . . . . . . . . . . . . .

40

Effect of cofactors Since it was possible t h a t extracts from glucose-grown cells were deficient in some cofactor(s) essential for the oxidation of the substrates tested, we e x a m i n e d the effect of NAD a n d F A D on the respiration of cell-free extracts in the presence of lactate, malate a n d succinate. As shown in Table IV, extracts from glucose-grown a n d from succinate-grown cells were both u n s a t u r a t e d in respect to NAD a n d the addition of the latter caused an increase in the rate of 02 u p t a k e in the presence of lactate a n d malate. However, one can readily see t h a t the activities of the two extracts were a u g m e n t e d b y addition of NAD to a p p r o x i m a t e l y the same e x t e n t ; the ratios between the respiratory activities of extracts from succinate-grown a n d glucose-grown cells remained within the range of 2 -3:1, as before the addition of cofactor. It is n o t e w o r t h v that addition of NAD resulted in considerable suppression of 0 2 u p t a k e in the presence of succinate. This finding is similar to t h a t of KEILIX AND HARTREE~s who showed that NAD inhibits succinate dehydrogenase, due to a c c u m u l a t i o n of oxaloacetate.

.4 ctivities of tricarboxvlic acid-cycle enzymes In view of the above findings we compared the activities of a n u m b e r of tricarboxvlic acid-cycle e n z y m e s in cell-free extracts from glucose-grown a n d succinate-

Biochim. Biophys. Acta, 93 (1064) 228-236

234

Y.s.

HALPERN,

A. E V E N - S H O S I - I A N , TABLE

E F F E C T O F C O F A C T O R S ON T H E R E S P I R A T I O N

IX"

OF CELL-FREE

G L U C O S E - G R O % V N E . c o l i IN T H E P R E S E N C E

[:or experimental

M. ARTMAN

EXTRACTS FROM SUCCINATE-

c o n d i t i o n s , see T a b l e I I1. Oxygen uptake c pl

.Substmtcs and cofa,ctors .

.

L-Malate L - M a l a t e + z o o ,ug N A D L - M a l a t e -4- - o o tzg N A I ) + 7 ° iLg F A D

.

AND

OF V A R I O U S S U B S T R A T E S

.5 mg extract protein of cells grc~,n on . . . . . . . . . . . Glucose .Succinatc

.

.

.

O~per 30 mtnj

.

.

2.5 mg extract protein of cells grmt,n on . . . . . Glucose N UCClnatc

32

09

o8

177

i0 4°

38 80

7°

-oo

4°

84

DL-Lactate

132

240

4°

120

D L - L a c t a t e ~- 2 o o t t g N A D Succinate S u c c i n a t e -i- 7 ° p g F A D Suecinate q Iopg FAD S u c e i n a t e -f 2 o o p g N A D

2io 168

35 ° 25255 250 IOO

89

ziS

1o7 I06 5t

I IO

21 I

~ 19 1IO 38

228 2I 7 89

grown cultures. The following enzyme activities were examined : acetate kinase, aconitate hydratase, isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase and malate dehydrogenase. The results of these experiments are summarized in Table V. One sees from these data that except for aconitase which displayed similar activities in both extracts, the activities of all the other enzymes tested were 2--3fold higher in extracts from succinate-grown cells than in those from glucose-grown cultures. TABLE

V

A C T I V I T I E S OF E N Z Y M E S OF T R I C A R B O X Y L I C ACID C Y C L E IN C E L L - F R E E

EXTRACTS

F R O M G L U C O S E - A N D S U C C I N A ' r E - G R O % V N i]. c o l i

T h e i i g u r e s in p a r e n t h e s e s d e s i g n a t e t h e n u m b e r o f e x p e r i m e n t s . .Specific actwtty ° Enzyrac

A c e t a t e kina.se

. . . . I n extracts f;om ~lucose- grown cells

3 . 5 " ::. 0 . 3 8

In extracts from succinate-grcal,n this

5 . 9 ° -- (>.3 °

(4) Aconitate

hydratase

5 8 . 0 0 .i , . o o

(4) lsocitrate dehydrogenase

4.3 ° -:. O.lO

(4) Succinate dehydrogenase

1.328.3o 210.OO

(4)

9 . 8 5 .! e . 7 5 3.3 o

! O. lO

(4) !_ 0 . 7 o

(4) Malate dehydrogenase

- 3.00

(4) ~ 0.08

(4) l"u m a r a t e h v d r a t a s e

61.oo (4)

97 .oo 2. 3.3 °

(4) 20.00

~J28.00 - 82.00

(4)

" Specific: a c t i v i t y is e x p r e s s e d a s u n i t s of e n z y m e p e r m g b a c t e r i a l p r o t e i n . B i o c h t m . Bwph.l's. d c t a , 93 (I9O4) 22S 2 3 0

SUCCINATE UTILIZATION BY GLUCOSE-GROWN

~ . coli

235

Concentration of intracellular succinate Experiments on the uptake of [a4Cisuccinate by glucose-grown and succinategrown cells were carried out as described in MATERIALSAND METHODS. The results are presented in Fig. 2. One can see that the concentration of succinate in the internal pool of cells incubated with [l*C]succinate was 3-4-fold higher in succinate-grown than in glucose-grown cells. In the presence of 20 ~moles/ml of succinate in the medium (roughly corresponding to the concentrations used in growth media) the internal concentration in succinate-grown and in glucose-grown cells was 35 and 9 t~moles/ml, respectively. 3~

t~ L

J

~

W

z_ u

S

C

SUCCINATECONCENTRATION~urnoles/rnlrnedLum

Fig. 2. Uptake of (14CJsuccinate by glucose-grown and succinate-grown E. coli. For experimental

details,

s e e MATIgRIALS AND METHODS.

DISCUSSION

An attempt has been made in these studies to elucidate some points concerning the inability of E. coli grown on glucose to utilize succinate as evidenced by the prolonged lag in growth upon transfer from a glucose to a succinate medium. In attempting to understand the reason for the inability of glucose-grown E. coli to grow on succinate as the sole source of carbon the following points were considered: I) glucose-grown cells lack an essential coenzyme or coenzymes involved in the oxidation of succinate; 2) glucose-grown cells lack tricarboxylic acid-cycle enzymes; 3) glucose-grown cells are impermeable to succinate. It became clear from our study that the inability of glucose-grown ceils to utilize succinate cannot be readily explained by the lack of a single factor involved in the oxidation of tricarboxylic acid-cycle intermediates, but is a result of an additive effect of several factors (low level of enzymes involved in the tricarboxylic acid cycle and low permeability to succinate). It has been shown that oxygen uptake by glucosegrown cells in the presence of succinate was about 9 °o of that of cells grown on succinate. On ttle other hand, the respiration of cell-free extracts prepared from glucosegrown cells in the presence of succinate was about one-half to one-third of that observed Biochim. Biophys. :Icta, 93 (z964) 228 .236

236

Y.s.

HAI.PERN,

A. E V E N - S H O S H A N ,

M. ARTMAN

with similar preparations from succinate-grown cells, thus indicating a permeability block in glucose-grown cells. Analysis of the levels of tricarboxylic acid-cycle enzymes and of the effect of the coenzymes NAD and FAD has next been attempted. The results showed clearly that the above coenzymes were limiting in both glucose- and succinate-grown cells to the same extent. The levels of all the tricarboxylic acid-cycle enzymes studied, with the notable exception of aconitase, in extracts from succinategrown cells were 2--3-fold higher than those in extracts from glucose-grown cells. These

data also suggested that the poor utilization of succinate by intact glucose-grown cells as compared with cell-free extracts (9 % v e r s u s 32-4 ° % of that obtained with similar preparations from succinate-grown cells) was due to a permeability barrier in glucose-grown cells to succinate. That permeability to succinate is an important factor in the utilization of the latter by microorganisms was also suggested by other authors 1~-16. Our results showed that succinate was taken up hv both glucose- and succinate-grown cells bv means of active transport. However, the concentration of succinate in glucose-grown cells incubated with succinate was only one-third to onefifth of that found in succinate-grown cells. It is clear that the combined effect of low activity of tricarboxvlic acid-cycle enzymes and the permeability block to succinate displayed by glucose-grown cells can explain the lag in growth observed upon transfer from glucose to a succinate medium. The general picture emerging from these experiments is one of partial repression by glucose of both the tricarboxvlic acid-cycle enzymes and the active transport of succinate.

ACKNOWLED(;EMENT This work was s u p p o r t e d in whole b y United S t a t e s Public Health Service Research ( ; r a n t GM 10044 a w a r d e d to one of us (Y.S.H.).

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Biochim. Biophys..-Iota, 93 (r9o4) z28-.23~,