BIOCHIMIE, 1971, 53, 1015-1021.

Studies on the transport of -methyl-D-glucoside in Bacillus subtilis 168.

Andr6 DELOBBE, Rosine HAGUENAUER and Georges RAPOPORT.

I n s t i t u t de B io log ie Moldcu la i re , CNRS , U n i v e r s i t d Par i s VII , 2, p lace duss i eu - - 7 5 - Par i s 5".

($-11-1971).

Summary . - - Some features of the uptake of ct-methyl-D-glueoside (aMG) by the PEP : glucose-phosphotransferase system were studied in B. subti l is 168. The transport system is inducible. Labeled products incorporated correspond to a mixture of free ctMG and (tMG-phosphate in equal amount. Whole intracellular aMG could be exchanged with external ctMG, which implies a continuous dephosphorylating process.

Experimental conditions for estimating the rate of ¢tMG uptake were improved. Washing the cells at 0°C causes within a few seconds leaking of 80 p. cent of the total labeling previously incorporated at 37°C. The cold treatment is non reversible, but loss of radio- activity could be avoided by washing the cells with a medium of high osmotic pressure.

The transport system of ctM(i in B. subli l is requires the presence of a carbon source .,;uch as glycerol or fructose.

INTRODUCTION.

The t ransport system of glucose has been exten- sively investigated in E s c h e r i c h i a col t and in S a l m o n e l l a t h y p h i m u r i u m (KESSLER and RICKEN- BERG [!l], HAGIHIRA, WILSON and LIN [2], HOEFEE, ENGLESBERG and LAMY [3I, GACHELIN [41, KABACK [5], HAGUENAUER and KEPES [6]).

Studies were car r ied out by using essentially a structural analogue of glucose, such as a-methyl- D-glucopyranoside, wh ich is metabolized only to ft-MG-6-phosphate.

The discovery of the PEP-sugar-phosphotrans- ferase system by KUNDIG and ROSEMAN [7], and its in v i t ro study have contr ibuted to the under- s tanding of the t ransport mechanism. PTS have been shown to be also involved in the uptake of sucrose, glucose or ctMG and fructose in B. sub t i l i s (LEPESANT and DEDONDER [8], MARQUET, WAGNER, DELOBBE, GAY and RAPOPORT [9!).

In the present study, we describe the t ransport and phosphory la t ion of aMG in more details, wi th par t icular at tention concerning the revers ibi l i ty of the t ransport process. Previous data by MAR- QUET and coll. [9] indicated that aMG-P was the unique product accumulated wi th in the ceils,

Abbreviat ions used : ctMG : a-methyl-D-glucoside. aMG-P : ct-methyl-D-glucose-6-phosphate. PEP : phospho-enol-pyruvate. PTS : PEP-hexose : phosphotransferase system.

suggesting a non-reversible mechanism. However , other exper imenta l condit ions, descr ibed below, allow the demonstra t ion of the concomitant accu- mulat ion of free (t~G and aMG-P.

The existence of the free form of aMG is in favor of the revers ibi l i ty of the t ransport system. Fur thermore , under these improved condit ions, the total amount of aMG incorpora ted into the cells was much higher than those estimated before.

MATERIAl, AND METHODS.

1 °) Uptake of labe led oMG.

B. sub t i l i s Marburg 168 was grown on mineral medium C, supplemented with 15 g/1 glycerol, at 37°C. (Jells in exponent ia l growth phase were harvested by centr i fugat ion at an O. D. of 1-1.5 (650 nm, d = 1 cm, spect rophotometer Beckman D. U), washed once wi th the medium Cl-glycerol and concentra ted in the same buffer. Bacterial suspensions were finally diluted to OD 0.5 (0.18 nag pro te in /ml ) in Cl-glycerol , in wh ich aMG (Calbio- chem) and methyl [a D-gluco 14C (U)] pyrano- side, specific act ivi ty 3.8 mci /mM, (Radiochemical Center) were added. 1 ml aliquots were w i t h d r a w n at intervals and filtered through Millipore mem- branes (HA 0.45 ~). Fil ters were r insed twice with 10 ml of Cl-glycerol at 37~C. After d ry ing the filters were counted in a toluene mixture wi th a scinti l lat ion spectrometer (Nuclear Chicago, Mark I).

I016 A ndrd Delobbe, Rosine Haguenauer and Georges Rapoporl.

2 °) Identification of accumulated intracellulm" products.

D e t e r m i n a t i o n of aMG-P b y t he b a r i u m b r o m i d e p r e c i p i t a t i o n t e c h n i q u e a c c o r d i n g to HORECK~.R a n d coll . [10], BUTTIN [!11]: t he t e s t ed s a m p l e (1 ml) w a s p i p e t t e d i n t o 9 ml 90 p. c e n t e t h a n o l (v /v ) c o n t a i n i n g 11 g/1 b a r i u m b r o m i d e . P r e c i - p i t a t e s w e r e f i l t e red on W h a t m a n G F / B glass f ibe r f i l ters a n d r i n s e d t w i c e w i t h 3 m l of 80 p. c e n t e t h a n o l . F i l t e r s w e r e d r i e d a n d t he r a d i o - a c t i v i t y e s t i m a t e d as m e n t i o n e d above .

K i n e t i c s of aMG u p t a k e w e r e p e r f o r m e d in five fold d i l u t e d m e d i u m (C2-glycero l ) to a v o i d p r e c i - p i t a t i o n of i n o r g a n i c p h o s p h a t e .

3 ° ) Media.

Medium C : (NH4),) S04, 2.5. 10 -2 M ; K2H P04, 7.10 -2 M ; KHoPO4, 3.10 -2 M ; MgSO4, 7 H20, 5.10 -4 M ; MnS04 , 4 H20, 1.10 -5 M ; ZnSO4, 7 H20, 5.10 -5 M ; f e r r i c a m m o n i u m - c i t r a t e 22 ~g/ml.

Medium C1 : (NH4) 2 S04, 2.5.10-2 M ; K2HP04, 7.10 -') M ; KH2P04, 3.10 -2 M ; c h l o r a m p h e n i c o l 200 t~g/ml.

Medium Cl-glycerol : C1 -4- g lyce ro l 10 g/1.

Medium C2 : (NH4) 2 S04, 0.5.10-2 M ; K2HP04,

CI No

50

~r r -

£9

B

0 Time r '~ 30

5O 17 °

J

0 Time mn 30

Fro. 1. - - Time course of uptake of z$C aMG : effect of different kinds of .washings.

B. subtilis 168 cells grown in C-glycerol 15 g/l , were induced wi th glucose 0.05 M for 45 minutes .

Incubat ion was carr ied out in Cl-glycerol 10 g/ l at 37 °, a t OD~o: 0.5, wi th aMG 10-4 M and 14C aMG 0.2 ixc/ml. Incorpora t ion of radioact ivi ty was followed by the Millipore f i l t ra t ion technique.

4 samples A, B, C and D of the same suspension were wi thdrawn, filtered and washed as follow :

- - sample A : wi th El-glycerol at 0 °, - - sample B : wi th CI-glycerol at 37 ° , - - sample C wi th El-glycerol 4- 1 M NaC1 at 0% - - sample D : wi th El-glycerol 4- 1 M NaC1 at 37 ° .

1.4.10 -2 M ; KH2POi, 0.6.10 -2 M ; c h l o r a m p h e n i c o l 200 ~ g / m l .

4 °) Definition of uptake unit.

One u n i t is de f i ned as t h e a m o u n t of the enzy- m a t i c t r a n s p o r t s y s t e m t h a t c a u s e d t he u p t a k e of 1 n a n o m o l e of s u b s t r a t e p e r m i n u t e , a t 37°C, at s a t u r a t i n g c o n c e n t r a t i o n (4.10-4 M).

R E S U L T S .

1 ° ) Total radioactivity incorporated and evi- dence for intracellular aMG.

D e t e r m i n a t i o n of s u g a r u p t a k e h a s b e e n r o u t i - ne ly d o n e , as yet, b y s a m p l i n g t he b a c t e r i a on Mi l l i po re f i l ters a n d w a s h i n g t he cel ls w i t h b u f f e r k e p t at 0 C (KEPES [12, 13]).

-g

~- 60 r -

"U

0

p

D

i

~" 0 T ime m n 60

FIe,. 2. - - K i n e t i c s of aMG uptake and phosphorglation. B. subtilis 168 was grown in C-glycerol 15 g/1 and

induced wi th glucose 0.05 M for 45 minutes . Incubat ion was performed at 37 °, in C2-glycerol

10 g/1 at OD~0 0.5 ; 14C aMG (0.1 l~e/ml) was added at a final concent ra t ion of 10-3 M.

At intervals , 3 samples A, B and C f rom the same batch were t reated as follows : (3 -- sample A : washing w i th Cl-glycerol a t 37 °, A sample B washing wi th Cl-glyeerol a t 0 °. × s a m p l e C : BaBr~ t r ea tmen t (see Material and

Methods).

W i t h B. subtilis 168, m u c h h i g h e r va lues w e r e o b t a i n e d b y ~ v a s h i n g t h e cel ls w i t h E l - g l y c e r o l at 37 °, o r w i t h C l - g l y c e r o l c o n t a i n i n g 1 M NaC1 at 0 ° (fig. 1). R a t i o s of i n i t i a l u p t a k e r a n g e d f r o m 2 to 5, d e p e n d i n g on t he e x p e r i m e n t .

N e w s t a n d a r d c o n d i t i o n s w e r e t h u s de f ined in w h i c h w a s h i n g t he b a c t e r i a w a s c a r r i e d out w i t h

BIOCHIMIE, 1971, 53, w' 9.

~-methylglucoside transport in B - s u b t H i s . 1017

m e d i u m C l - g l y c e r o l p r e w a r m e d at 37 ° . S u c h c o n d i t i o n s a p p e a r e d also va l i d fo r t he d e t e r m i - n a t i o n of g lucose a n d f r u c t o s e u p t a k e in B. sub- tilts. W a s h i n g w i t h C l - g l y c e r o l a t 37 ° led to va- lues 2 to 3 t i m e s h i g h e r t h a n t h o s e e s t i m a t e d a f t e r w a s h i n g t he cel ls w i t h co ld C l - g l y c e r o l .

T h e p r e c i p i t a t i o n of ~MG-P by t he b a r i u m bro - m i d e t e c h n i q u e a l l o w e d fas t a n d a c c u r a t e d k i n e t i c s . T h e t i m e c o u r s e of s u c h a n e s t i m a t i o n is s h o w n in fig. 2, c o m p a r e d to t he to ta l r a d i o - a c t i v i t y i n c o r p o r a t e d a f t e r w a s h i n g at 0 ° a n d 37 ° r e s p e c t i v e l y .

F r e e aMG a c c o u n t e d for 40 to 50 p. c en t of t he to ta l l a b e l i n g a c c u m u l a t e d w i t h i n t he cells . W a s h i n g w i t h co ld b u f f e r led to a v a l u e of to t a l r a d i o a c t i v i t y l o w e r t h a n t h o s e c o r r e s p o n d i n g to ~MG-P, a n d it w a s c o n c l u d e d t h a t p a r t of t he p h o s p h a t e d e r i v a t i v e w a s lost f r o m t he cells.

T h e loss of r a d i o a c t i v i t y a f t e r w a s h i n g at 0 ° was s t u d i e d in a d i f f e r e n t w a y . To i n d u c e d ce l l s of B. subtilis, e q u i l i b r a t e d at 37 °, l a b e l e d aMG w a s a d d e d (see M a t e r i a l a n d M e t h o d s ) . Af t e r 7 m i n u t e s , t he s u s p e n s i o n w a s r a p i d l y c h i l l e d to 3 ° ( w i t h i n 45 s e c o n d s ) a n d t h e s u g a r u p t a k e w a s d e t e r m i n e d by t he Mi l l i po re f i l t r a t i o n t e c h n i q u e a n d w a s h i n g w i t h C l - g l y c e r o l at 37 °. T h e r e s u l t s s h o w e d t h a t t he b a c t e r i a a l l o w e d to s tay at 3 ° c o n t a i n e d o n l y one f i f th to one f o u r t h of t he r a d i o - a c t i v i t y i n c o r p o r a t e d at 37 ° . T h i s v a l u e r e m a i n e d c o n s t a n t as l ong as t he cel ls w e r e i n c u b a t e d at 3 ° ; t h e s a m e r e s u l t w a s o b s e r v e d w h e n t he m i x t u r e w a s c h i l l e d to 3 ° fo r 8 m i n u t e s a n d w a r m e d up a g a i n at 37 ° . T h e cel ls d i d no t r e c o v e r t h e i r i n i t i a l ac t iv i ty , s h o w i n g t h a t t he << co ld s h o c k >> effec t w a s n o n r e v e r s i b l e . C h a n g e in t h e O D w a s t e s t ed d u r i n g t h i s t r e a t m e n t : a d e c r e a s e of less t h a n 10 p. c en t of t he i n i t i a l va lue w a s f o u n d at t he end , s u g g e s t i n g t h a t the << co ld s h o c k >> w a s u n l i k e l y to cause i m p o r t a n t lys i s of t he bac t e r i a .

2 ° ) Induction, differential rate of synthesis, apparent affinity of the transport system.

T h e t r a n s p o r t s y s t e m of uMG is i n d u c i b l e , a n d the i n d u c t i o n c o u l d be a c h i e v e d e i t h e r b y g lucose o r b y aMG [9].

T h e d i f f e r e n t i a l r a t e of s y n t h e s i s , d e t e r m i n e d w i t h g r o w i n g b a c t e r i a in m e d i u m C-g lyce ro l 15 g / l , w a s 160 u n i t s p e r a n i n c r e a s e of t h e b a c t e - r i a l p r o t e i n s of 1 my, a t 37 ° , w h e n s a t u r a t i n g c o n c e n t r a t i o n of i n d u c e r w a s e m p l o y e d ( a b o v e 10 -4 M).

T h e v a l u e m e a s u r e d for u n i n d u c e d cel ls w a s less t h a n o n e h u n d r e d t h .

1 V

1.0

0.5

I M -I I C~ ocMG

Fla. 3. - - Lin~u, eaver-Burk plot for the determination of K,~ of the transport system for c~MG.

Culture of B. subtilis 168 in C-glycerol 15 g/1 was induced for 120 minutes wi th glucose 0.05 M.

Abscissa : reciprocal values of molar concentra t ions of uMG.

Ordinate : reciprocal values of the in i t ia l rate of uMG uptake expressed as n m o l e s / m i n u t e / m g of bac- ter ial proteins.

TABLE I.

Characteristics of the transport systems of aMG, glucose and fructose.

Different ia l rate of s y n t h e s i s (') :

- un induced culture (units) . . . . . . . . . . . . . . . . . . - induced culture (") (units) . . . . . . . . . . . . . . . . . .

Half-maximum induction (M) . . . . . . . . . . . . . . . . . . . app. Km of uptake (M) . . . . . . . . . . . . . . . . . . . . . . . . .

aMG G l u c o s e

I 0.9 l 0.9 160 170

2. 10 -'~ 3. 10 -s 4. 10-;' 1. 10 -'~'

Fructose

0 .9 130

2. 10-:'

( ' ) See definition of uni t in Material and Methods. ( '*) Measured at sa tura t ing concentra t ion of indueer. B. subtilis 168 was grown at 37 ° in medium C supplemented with glycerol

15 g/1.

BIOCH1MIE, 1971, 53, n ° 9.

1018 Andr~ Delobbe, Ros ine H a g u e n a u e r and Georges Rapopor t .

S i m i l a r r e su l t s w e r e o b t a i n e d in t he case of g lucose a n d f r u c t o s e ( T a b l e I).

T h e c o n c e n t r a t i o n of i n d u c e r l e a d i n g to ha l f - m a x i m a l i n d u c t i o n w a s , in t h e s a m e c o n d i t i o n s , 3.10 -5 M a n d 2.10 -.~ M for g lucose a n d aMG r e s p e c - t ive ly .

T h e a p p a r e n t a f f in i ty of t he t r a n s p o r t s y s t e m fo r aMG w a s f o u n d to b e 4.10-~ M (fig. 3), w h i c h was c lose ly s i m i l a r to t h o s e m e a s u r e d f o r g lucose a n d f ruc tose .

3 ° ) Exchange reaction.

T h e a c c u m u l a t e d i n t r a c e l l u l a r p r o d u c t s of aMG a re e n t i r e l y e x c h a n g e a b l e w i t h e x t e r n a l aMG : t h i s w a s e s t a b l i s h e d b y a d d i n g , , a t i n t e r v a l s , 14C aMG to a b a c t e r i a l s u s p e n s i o n p r e l o a d e d at z e ro t i m e w i t h n o n - l a b e l e d aMG. T h e u p t a k e of r a d i o a c t i - v i ty , s h o w n in fig. 4, r e a c h e d a m a x i m u m value , f o l l o w e d b y a s l o w d o w n c o r r e s p o n d i n g to an eff lux p h e n o m e n o n .

J 0

o

t -

t~ D

i

0 S0 Time mn 100

FI(;. 4. - - - Kinetics of ~MG exchange. B. subtilis 168 cells grown in C-glycerol 15 g/i, were

induced wi th ghicose 0.05 M for 120 minutes . Kinetics were performed in Cl-glycerol, at 37 °, at OD~o 0,5 wi th ¢tMG 4.10-4 M.

z4C aM,G (0.03 ~c /ml) was added at zero time, 15 miuutes, 30 minutes and at 45 minutes .

Lys i s of t he b a c t e r i a w a s r u l e d out b y t h e f o l l o w i n g e x p e r i m e n t : i n d u c e d cel ls w e r e w a s h e d a n d i n c u b a t e d in m e d i u m C1 c o n t a i n i n g g lyce ro l a n d c h l o r a m p h e n i c o l , for 30 m i n u t e s at 37 °. T h e s e ce l l s a c c u m u l a t e d aMG at t h e s a m e r a t e at t h o s e r e c e i v i n g aMG i m m e d i a t e l y a f t e r w a s h i n g .

E x i s t e n c e of an e x c h a n g e r e a c t i o n i n v o l v e d s i m u l t a n e o u s in f lux a n d eff lux p r o c e s s e s : w h e n

the m a x i m a l u p t a k e is r e a c h e d , t h e eff lux m e c h a - n i s m b e c o m e s p r e p o n d e r a n t .

T h e i n i t i a l r a t e of r e n e w a l w a s f o u n d to be l o w e r t h a n t he i n i t i a l r a t e of u p t a k e , a n d it d e c r e a s e d w i t h t i m e : a f t e r 15 m i n u t e s of p r e - l oad ing , i t a c c o u n t e d fo r 40 p. c e n t of t h e i n i t i a l r a t e of u p t a k e a n d a f t e r 30 m i n u t e s fo r 20 p. c en t on ly .

4 ° ) Effects of sodium azide and sodium fluoride.

I t h a s b e e n s h o w n in E. colt [6] t h a t t he j o i n t a c t i o n of t he t w o c o m p o u n d s i n d u c e s t he eff lux of all f ree uMG p r e v i o u s l y a c c u m u l a t e d , l e a v i n g

E

5"

c- o

o >. . t 0

-8 0 t " Q..

" 0 t - O

v p O .

o Ig i s

40 NaF+Az

V "

0 Time mn 30

FIfi. 5. - - Combined action of NuNs and NaF on the transport and phosphorylation of aMG

B. snbtilis 168 was grown on C-glycerol 15 g/l, induced wi th glucose 0.05 M for 45 minutes .

Incorpora t ion was followed in C2-glycerol 10 g/ l , at 37 °, at OD~o 0.5 (aMG 10-4 M ; 14C aMG 0.1 ~c/ml) .

2 al iquots f rom the same mix ture were w i thd rawn : - - al iquot A : total radioact ivi ty. - - al iquot B : radioact ivi ty precipi table by BaBr=. At 8 minutes , a f rac t ion of the control suspension

(I) was taken up and incubated wi th NaNa 4.10-2 M ÷ NaF 5.10-2 M (II).

u n c h a n g e d t he leve l of i n t e r n a l (~MGP : a f t e r 15 to 20 m i n u t e s of r e a c t i o n t he t o t a l a m o u n t of s u g a r a c c u m u l a t e d equa l s t he a m o u n t of t he p h o s - p h o r y l a t e d d e r i v a t i v e . T h i s w a s s h o w n to be a c o n s e q u e n c e of s i m u l t a n e o u s i n h i b i t i o n of p h o s - p h o r y l a t i o n a n d d e p h o s p h o r y l a t i o n b y t h e s e t w o c o m p o u n d s .

A s i m i l a r r e s u l t w a s o b t a i n e d w i t h B. subtilis i n p r e s e n c e of az ide a n d NaF , a d d e d w h e n t h e u p t a k e r e a c h e d i ts m a x i m a l va lue .

No effec t w a s d e t e c t e d on t he t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d o r on t h e level of aMG-P, w h e n az ide a n d N a F w e r e a d d e d s e p a r e t e l y .

BIOCHIMIE, 1971, 53, n ° 9.

a - m e t h y l g l u c o s i d e t r a n s p o r t in B - s u b t i l i s . 11)t9

5 °) R e q u i r e m e n t of a carbon source.

B. subt i l is 168 ce l l s d e p r i v e d of g lyce ro l or of a n y o t h e r c a r b o n s o u r c e w e r e p r e p a r e d f r o m a c u l t u r e g r o w n on m e d i u m C + g lyce ro l 15 g / l , a n d i n d u c e d fo r 2 h o u r s w i t h g lucose 0.05 M. Af te r c e n t r i f u g a t i o n a n d w a s h i n g w i t h m e d i u m C1, the b a c t e r i a w e r e r e s u s p e n d e d in the s ame b u f f e r (fig. 6).

A f i rs t s a m p l e w a s w i t h d r a w n a n d r e c e i v e d i m m e d i a t e l y g l y c e r o l a n d 14C aMG ( s u s p e n s i o n A). A s e c o n d one w a s i n c u b a t e d at 37" a n d r e c e i v e d the l abe led c o m p o u n d a f t e r 20 m i n u t e s ( su spen - s ion B).

A p o r t i o n f r o m t he s u s p e n s i o n B w a s t a k e n up 10 n f i n u t e s a f t e r t he a d d i t i o n of 14C aMG a n d w a s s u p p l e m e n t e d w i t h g lyce ro l ( s u s p e n s i o n C). A las t s a m p l e w a s i n c u b a t e d at 37 ° fo r 30 m i n u t e s b e f o r e r e c e i v i n g b o t h g lyce ro l a n d 14C uMG ( s u s p e n s i o n D).

40.

r

o O. D

o :E

QA

GlY \t ~MG~f

0

~ D

,,tMG + Glyc i

Time mn 60

Fro. 6. - - Effects of glycerol as a carbon som ce on the uptake of ctMG.

B. subtilis cells were grown in C-glycerol 15 g/ l Induced wi th glucose 0.05 M for 120 minutes , and washed wi th medium C1.

Incorpora t ion was carried out in the same buffer at 37 °, at OD~0 0.5. Suspension A : at zero time, addi t ion of aMG 10-4 M

+ 14C aMG 0,03 ~c /ml and glycerol 10 g/l .

Suspension B : at 20 minutes , addi t ion of c{MG 10-4 M + 14C aMG 0.03 ue/ml .

Suspension C : at 30 minutes , addi t ion of glycerol 10 g/1 to a sample f rom suspension B.

Suspension I) : at 30 minutes, addi t ion of aMG 10-4 M -~ 14C ctMG 0.03 g c / m l and glycerol 10 g/1.

Resu l t s p r e s e n t e d in fig. 6 i n d i c a t e d tha t , in a b s e n c e of c a r b o n sou rce , t he i n i t i a l u p t a k e w a s 8 t i m e s l o w e r t h a n in p r e s e n c e of g lyce ro l , and t h a t t he m a x i m u m level r e a c h e d w a s also l o w e r ( a b o u t 6 t imes ) .

6 0

C

,¢

o O-

L9 7r

s A

! ~ C

F ~ u

T~ne mn 60

Fro. 7. - - Effect of fructose as a carbon source on the uptake of aMG.

B. subtilis was grown in medium C + fructose 15 g/l , induced wi th glucose 0.05 M for 120 minutes and washed w i th medium C1; incubat ion was done in medium El, a t 37 °, at OD,~w 0.5. Suspension A : at zero time, addi t ion of aMG 10-4 M

+ 14C aMG 0.03 ~e /ml + fructose 10 g/1 ;

Suspension B : at 20 minutes , addi t ion of ¢tMG 10-4 M + 14C ctMG 0.03 :~e/ml ;

Suspension C : at 30 minutes , addi t ion of fructose 10 g/1 to a sample derived from suspension B.

Moreove r , u n d e r s t a r v a t i o n c o n d i t i o n s t he to ta l a m o u n t of r a d i o a c t i v i t y w i t h i n the cel ls r e m a i n e d c o n s t a n t ( c u r v e B). A d d i t i o n of g lyce ro l at 30 mi- nutes , b e f o r e r e a c h i n g t he p l a t eau , c a u s e d on ly a s low i n c r e a s e in the r a t e of u p t a k e ( c u r v e C). T h e va lue o b t a i n e d in the l a t t e r case was fa r l o w e r t h a n t h o s e of a c o n t r o l in w h i c h g lyce ro l a n d ctMG w e r e p u t t o g e t h e r a f t e r 30 m i n u t e s of i ncu - b a t i o n ( c u r v e D). In e x p e r i m e n t C, t he i n i t i a l r a t e r e p r e s e n t e d 10 p. c en t of t he ve loc i t y m e a s u r e d in e x p e r i m e n t D ( in o t h e r e x p e r i m e n t s i t a c c o u n - ted for 25 p. cen t ) .

To see w h e t h e r o r - n o t t h i s p h e n o m e n o n w a s specif ic , a n o t h e r c a r b o n s o u r c e was t e s t ed : f ruc - tose w a s u sed in p l ace of g lycero l . B. subt i l is 168 was g r o w n on m e d i u m C s u p p l e m e n t e d w i t b f r u c t o s e 15 g/1 a n d i n d u c e d for 2 h o u r s w i t h glu- cose 0.05 M. B a c t e r i a l m i x t u r e s w e r e p r e p a r e d as d e s c r i b e d above . T h e r e su l t s s h o w n in fig. 7 w e r e

BIOCHIMIE, 1971, 53, n ° 9.

1020 Andrd Delobbe, Rosine Haguenauer and Georges Rapoporl.

similar to those already observed in the case of glycerol, the unique measurable difference con- cerned the rate of uptake upon addi t ion of fruc- tose (curve C) wh ich was the same as in the control exper iment (curve A). No substantial variat ion was found in the affinity of the trans- port system for aMG, under starvation condit ions (apparent Km ~ 2.10 -5 M).

DISCUSSION.

The s tandard condi t ions for est imating sugar incorpora t ion have been modified. Cells are not lysed by washing at 0 ° but it is l ikely that the permeabi l i ty of the bacter ia l membrane is increased by the treatment. After 8 minutes at 3 ° the accumulat ion process is i r revers ib ly impaired. This can be avoided if the tempera ture of the washing buffer is not changed or if the washing at 0 ° is made with a high osmotic pressure medium. Closely s imilar results were described by LEDEn and PERRY [14] wi th several strains of E. colt K 12: the rate of galactose, valine and thiomethyl-D-galactoside accumulat ion depends upon w a s h i n g : washing at 0 ° gives rise to a extremely rapid loss (5 to 10 seconds) of 90 p. cent of the substrate ; this loss is avoided if the r ins ing fluid is mainta ined at the tempera ture of the incubat ion mixture or, if the washing medium at 0 ° has a higher osmotic pressure. Substrates studied by LEDER and PEnnY are accumulated by permeases. In B. subtilis 168, the carbohydrates studied in the present work are t ranspor ted by PTS and loss of both in t racel lu lar free aMG and aMG-P occurs dur ing cold shock.

ENGLESBERG and NOVOTNY [15] observed a << cold shock >> effect dur ing accumulat ion of L arabinose by the permease system of E. colt B/r. When the cells previously loaded at 37 ° wi th arabinose are chil led to 0 °, an efflux of 75 p. cent of the total sugar accumulated is observed wi th in a few seconds. But in this case, the << cold shock is ent i rely r e v e r s i b l e : L arabinose is taken up again to the same level, when the suspension is warmed u I) from 0 ° to 37 ° .

Recently, KEPES made a cr i t ical survey of the different methods employed in t ransport studies [13I. He stressed out that the cold shock varies from one strain to another and the difference of osmotic pressure between the incubat ion mixture and the r insing medium is very i m p o r t a n t : washing wi th hypo-osmotic fluid produced wi th in 20-30 seconds of filtration loss of 50 p. cent or more of the pool.

Contrary to other bacteria, the t ransport system of glucose is inducible in B. subtilis 168. This finding was also reported by FREESE and coll. [l~i].

The t ime course of aMG uptake presents a par t icular feature because no real steady-state is reached. Decrease in the total amount of accumu- lated sugar implies an exit process and is con- sistant wi th a reversible t ransport mechanism. Exchange react ions provide evidence for a con- comitant influx and efflux process. In the first 20 minutes the rate of uptake is predominant , then it slows down and becomes lower than the efflux rate.

Lysis of the cells could be excluded. The de- crease observed in the exchange veloci ty dur ing incubation reveals a gradual s lowdown in the influx rate of uptake. This can be due to the intra- cellular accumulat ion of a-MG-P. Such an inhibi- t ion by phosphate sugars was already postulated by KAEACK [!17] for aMG transport and phospho- rylat ion by PTS from membrane preparat ions.

After combined action of azide and NaF the amount of ctMG-P remains constant and equals the total intracel lular sugar level, indicat ing that only free alVfG is lost from the ceils.

As phosphoryla t ion must be inhibi ted in those condit ions, this can be in terpre ted by a simulta- neous inhibi t ion of the dephosphoryla t ion step.

This is another indicat ion that after phospho- rylat ion by PTS, aMGP is usually dephosphory- lated, only free (tMG being capable of exit from the ceils.

In any case, to explain the exchange react ion a continuous dephosphoryla t ing process must be taken into account : data obtained in the present study are very similar to those descr ibed before by HAGUENAUER and KEPES [6]. Several phospha- rases have been already descr ibed in B. subtilis [18], and it is possible to attribute a key role to one of them in the dephosphoryla t ing step of in t racel lular aMG-P.

Requi rement of a carbon source in order to reach the maximal rate of incorpora t ion is con- t rary to the results reported in E. colt by HAGIrlIRA and coll. [2] and by KESSLER and RICKENBERG [1] who showed that glycerol is a compet i t ive inhi- bitor of aMG uptake.

In B. subtilis, the need of a carbone source is almost absolute. It gives rise to a pool of high- energy metabolites, of w h i c h PEP is the main component , and wh ich are essential for the wor- king of the t ransport system. Under starvation

BIOCHIMIE, 1971, 53, n ° 9.

u - m e t h y l g l u c o s i d e t r a n s p o r t in B-sub t i , l i s . 1021

c o n d i t i o n s , the pool b e c o m e s r a p i d l y e x h a u s t e d , and ref i l led aga in w h e n the m e d i u m is s u p p l e m e n - ted w i t h an e x t e r n a l c a r b o n source . R e c o n s t i t u t i o n of the poo l d e p e n d s upon tbe na tu re of the c a r b o n c o m p o u n d . W i t h g lycero l , the pool of P E P is res - t o r e d on ly if it is a d d e d t o g e t h e r w i t h ¢tMG. W h e n aMG is a d d e d in the dep l e t ed m e d i u m be fo re gly- cero l , t hus l e ad ing to the a c c u m u l a t i o n of a-MG-P, f u r t h e r a d d i t i o n of th is c a r b o n sou rce does not c h a n g e s ign i f i can t ly the up take of aMG. One can a s sume tha t aMG-P i n t e r f e r e s w i t h the a c c u m t d a - t ion or the m e t a b o l i s m of g lyce ro l a n d r e d u c e s the P E P syn thes i s . Such an i n t e r a c t i o n w o u l d not o c c u r in the case of f ruc tose .

A cknawledgment.

This work was supported by a grant from the <~ D6- par tement des Mol6cules Marqu6es >) (Commissariat h l 'Energie Atomique).

R~SUM~.

L'accumulation de l 'alpha-m6thyl-D-glucoside (aMG) par le syst6me phospho-6nol-pyruvate-D-glucose-phos- photransf6rase a 6t5 dtudi6e chez B. subtilis 168. Le syst6me de I~n6tration est inductible.

Les produits d'accumulation, analysds h l 'aide d'nMG radioaetif, correspondent h u n m61ange d'aMG libre et d'aMG-6-phosphate, en quantit6 fiquivalente. L'nMG intracellulaire est enti6rement dchangeable avec de I'aMG externe, ce qui implique un processus continu de d6phosphorylation.

Les conditions de mesure de la vitesse de p6n6tra- tion ont ~t6 modifi~es. Le lavage h 0 ° des bact6ries provoque en quelques secondes uu chassage de 80 p. cent de la radioactivit6 totale pr6alablement accu- mul6e h 37°C. Ce chassage est irr6versible, et peut ~tre ~vit6 si le lavage est r6alis6 avec un milieu de forte pression osmotique.

Le syst6me d'accumulation de I'aMG par B. subtilis ne pent fonctionner qu'en pr6sence d 'une source de carbonc, tel le glyc6rol ou le fructose.

ZUSAMMENFASSUNG.

Die Anhiiufung des alpha-Methyl-D-Glukosids (aMG) durch das Phosphoenol-Pyruvat : D-Glukose-Phospho-

t ransferase-System ist bei Bacillus subfilis 168 unter- sucht worden. Das Zutri t tsystem ist induzierbar.

Die Anh/iufungsprodukte, die mittels radioktivem ctMG analysiert wurden, entsprechen einer Mischung von freiem ctMG und von aMG-6-Phosphat in gleicher Menge. Das intrazelluliire aMG kann gegen das ~iussere uMG vollsti~ndig vertauscht werden, was auf ein dauerndes Dephosphoryl ierungsverfahren zurfickzu- fiihren ist.

Die Messungsbedingungen der Zutri t tgeschwindigkeit sind geiindert worden. Waschen der Bakterien bei 0 ° verursacht in einigen Sekunden ein Vertreiben von 80 p. cent der gesamten Radioaktivit~it, welche vorher bei 37 ° angeh/iuft worden war. Dieses Vertreiben ist irreversibel und kann vermieden werden, wenn das Waschen in einem Medium yon starkem osmotischen Druck erfolgt.

Das Anh~ufungssystem des ctMG bei B. subtilis kann nur in der Gegenwart einer Kohlenstoffquelle, wie Glycerol oder Fruktose funktionieren.

REFERENCES.

1. KESSLBR, D. P. and RICKENBERG, U. V., Biochem. Biophys. Res. Com., 1963, 10, 482.

2. HAGIHIRA, H., WILSON, T. H. and LIN, E. C. C., Biochim. Biophys. Acta, 1963, 78, 505.

3. HOFFEE, P., ENGLESBERG, E. and LAMV, F., Biochim. Biophys. Acta, 1964, 79, 337.

4. GACHELIN, G., Eur. J. Biochem., 1970, 16, 342. 5. KABACK, H. R., J. Biol. Chem., 1968, 243, 3711. 6. HAGUENAUER, R. and KEPEs, A., Biochimie, 1971,

5:3, 99. 7. KUNDm, W. and ROSEMAN, S., J. Biol. Chem., 1971,

246, 1393 ; 1407. 8. LEPESANT, J.-A. and DEDONDER, R., C. R. Acad. Sc.,

1968, 267 D, 1109. 9. MARQUET, M., WAGNER, M.-C., DELOBBE, A., GAY, P.

and RAPOPORT, G., C. R. Acad. Sc., 1970, 271 D, 449.

10. HonECKER, B. L., THOMAS, J. and MONOR, J., J. Biol. Chem., 1960, 235, 1580.

11. BUTTIN, (i., J. Mol. Biol., 1963, 7, 164. 12. KEPES, A., Biochim. Biophys. Acta, 1960, 40, 70. 13. KEPES, A., J. Membrane Biol., 1971, 4, 87. 14. LEDEn, I. G. and PEnnY, J. W., Fed. Proced., 1967,

26, 394. 15. ENGLESBERG, E. and NOVOTNY, C. P., Biochim. Bio-

phys. Acta, 1966, 117, 217. 16. FREESE, E., KLOFAT, W. and GALLIERS, E., Biochim.

Biophys. Aela, 1970, 222, 265. 17. KABACK, H. R., Proc. Nat. Acad. Sc., U. S., 1969,

63, 724. 18. REMAIN, A. L. and HENDLIN, D., J. Bact., 1967, 94,

66.

BIOCHIMIE, 1971, 53, n ° 9.