Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis : constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168

BIOCHIMIE, 1977, 59, 287-292.

Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168. F r a n k KUNST, Michel STEINMETZ, J e a n - A n t o i n e LEPESANT a n d R a y m o n d DEDONDER ~.

Ddpartement de Biochimie Cellnlaire, Inslital de Recherche en Biologie MoIdcMaire, C.N.R.S. et Universitd Paris VII, Toar 43, 2 place Jussieu, 75221 Paris Cedex 05 (France). (76-11-1976). Summary. - - A .~-D-fructofuranosidase - - called levanase - - capable of the hydrolysis of sucrose, inulin and /evans has been identified in Bacillus sublilis Marburg. This enzyme call not be detected in strain 168. Ho'wever, sacL mutations - - mapped on the chromosome of strain 168 between the pheA and aroD reference markers - - lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose. INTRODUCTION. T w o sucrose i n d u c i b l e enzymes i n v o l v e d in sucrose h y d r o l y s i s h a v e a l r e a d y been c h a r a c t e r i z e d in Bacillus sabtiIis M a r b u r g [1, 2, 33 : an extracellular l e v a n s u c r a s e and an i n t r a c e l l u l a r sucrase. L e p e s a n t el al. [4, 5] started a genetic analysis of the r e g u l a t i o n of the syntheses of these e n z y m e s i n v o l v i n g m a p p i n g of m u t a t i o n s by t r a n s f o r m a t i o n and PBS1 t r a n s d u c t i o n . T h r e e types of constitutive m u t a t i o n s h a v e been d i s t i n g u i s h e d : mutations l e a d i n g to c o n s t i t u t i v e l e v a n s u c r a s e synthesis (sacR), c o n s t i t u t i v e sucrase synthesis (sacT), and c o n s t i t u t i v e synthesis of both sucrase a n d l e v a n s u c r a s e (sacS). A study of sacS a n d sacT m u t a n t s g r o w n in mineral media supplemented with carbon sources such as glucose, g l y c e r o l or sucrose r e v e a l e d that the rate of c o n s t i t u t i v e sucrase synthesis w a s l o w because of catabolite r e p r e s s i o n . D u r i n g g r o w t h w i t h glutamate + s u c c i n a t e as the c a r b o n sources, release f r o m r e p r e s s i o n allows a h i g h e r rate of synthesis [3]. Constitutive sacR, sacs and sacT m u t a n t s h a v e been isolated on solid m i n e r a l g l y c e r o l m e d i u m (after s c r e e n i n g for the p h e n o t y p e <>) w i t h the use of a c o l o u r test [4]. In v i e w of the r e p r e s s i o n e x e r t e d by g l y c e r o l w e d e c i d e d to isoIate a d d i t i o n a l m u t a n t s a p p l y i n g the same m e t h o d but on solid m i n e r a l m e d i u m s u p p l e m e n t e d w i t h glutamate + citrate as the carbon sources. O To whom all correspondence should be addressed.

On this m e d i u m w e isolated a n e w class of inurants (sacL) s y n t h e t i z i n g c o n s t i t u t i v e l y a different sucrose h y d r o l y z i n g enzyme, called levanase. MATERIALS AND METHODS. CHEMICALS.

I n u l i n ( m o l e c u l a r w e i g h t ~ 5000) was p u r c h a sed f r o m Merck. Levans w e r e p r e p a r e d in this l a b o r a t o r y and h o m o g e n i z e d w i t h r e s p e c t to molec u l a r w e i g h t (mean value : 15000) using a c o l u m n of Biogel P30 (Biorad). BACTERIAL STRAINS.

The strains table I.

used in this study are listed in

MEDIA.

C m e d i u m c o n t a i n i n g m i n e r a l salts and MM med i u m c o n t a i n i n g m i n e r a l salts and citrate h a v e been p r e v i o u s l y d e s c r i b e d [1, 4]. F o r l i q u i d cultures, C m e d i u m was s u p p l e m e n t e d w i t h t r y p t o p h a n (100 mg/1) and c a r b o n sources : p o t a s s i u m glutamate (0.07 M) + p o t a s s i u m s u c c i n a t e (0.07 M), or glucose (10 g/l) or g l y c e r o l (10 g/l) or sucrose (10 g/1). MM-glycerol and MM-glutamate plates c o n t a i n MM solid m e d i u m s u p p l e m e n t e d t r y p t o p h a n (20 mg/1), e v e n t u a l l y o t h e r a u x o t r o p h i c r e q u i r e m e n t s (20 m g / l ) and e i t h e r g l y c e r o l (1 g / l ) or potassium glutamate (7 ruM) as c a r b o n sources. ISOLATION OF sacL MUTANTS.

E t h y l m e t h a n e sulfonate (EMS) m u t a g e n i z e d cultures of strain 168 [4] ~vere d i l u t e d and plated on MM-glutamate solid m e d i u m . After i n c u b a t i o n at

F. K u n s t a n d coll.

288

37°C separate colonies a p p e a r e d . A c o l o u r test based on the d e t e c t i o n of the glucose l i b e r a t e d after sucrose h y d r o l y s i s has been a p p l i e d as des-

or r e c o m b i n a t i o n b e t w e e n sacL and aroD, pheA or leuA m a r k e r s w e r e c a l c u l a t e d as p r e v i o u s l y d e s c r i b e d [41.

TABLE I.

List of strains. Strain

6enotype

Origin of derivation

168 GSY225 SB120 QB25 QB33 QB35 QB36 QB39

C. Anagnostopoulos

QB46 QB101 QB127 QB136 QB166 QB167 QB168 QB169 QB171

trpC2 pheA1 trpC2 aroD120 lrpC2 sacSc49 trpC2 sacScSO trpC2 sacRc44 trpC2 sacRc37 lrpC2 sacTc30 lrpC2 sacRc41 lrpC2 sacScg8 trpC2 melC3 leuA8 lrpC2 sacUh200 leuA8 trpC2 sacUh32 leuA8 lrpC2 sacL5 lrpC2 sacL6 trpC2 sacL7 trpC2 sacL8 lrpC2 sacL12 lrpC2

QB666

lhr5 leuA8 hisA1 sacA321

QB2018

sacL6 leuA8 hisA1 sacA321

QB2019

sacL6 sacUh200 leuA8 sacA321

QB43

QB2020 QB2021

B

N. Harford Lepesant et al. [4] m

i

Kunst el al. [81 EMS on 168

markers introduced into 168 by transformation in several steps tf (b) QB167 ~ QB666

tf QB127

~" QB2018 tf

sacL6 sacUh32 leuA8 sacA321

QB136

~- QB2018 tf

sacL6 leuA8 sacA321

QB127

~" QB2018

(b) Strains constructed by transformation.

c r i b e d by L e p e s a n t et al. [4]. Colonies of strain 168 r e n l a i n u n c o l o u r e d and r e d halos a p p e a r a r o u n d clones of c o n s t i t u t i v e mutants. T h e mutants w e r e p i c k e d up and reisolated. In this way, several sacL m u t a n t s h a v e been isolated i n d e p e n dently. PBS1 TRANSDUCTION. The t r a n s d u c f i o n p r o c e d u r e has been d e s c r i b e d e a r l i e r [4!. PBS1 lysates of sacL mutants h a v e been utilized to t r a n s d u c e the r e c i p i e n t s SBI20, QB'101 or GSY225 (table I). Aro +, Leu + or Phe + rec o n l b i n a n t s h a v e been selected on MM-glycerol m e d i u m , reisolated, s t r e a k e d as p a t c h e s on the same m e d i u m a n d r e p l i c a p l a t e d on MM-glutamate m e d i u m . Using the c o l o u r test on the latter med i u m r e c o l n b i n a n t s ~xith a c o n s t i t u t i v e p h e n o t y p e c o u l d be identified. The p e r c e n t a g e s of c o t r a n s f e r

BIOCH1MIE, 1977, 59, n ° 3.

QUANTITATIVE

DETERMINATION

OF

LEVANASE

ACTI-

VIT'Y.

Levanase a c t i v i t y can be n l e a s u r e d as the rate of h y d r o l y s i s of i n u l i n (~-l,2-fructan), sucrose or levan (~-2,6-fructan). Inulin, w h i c h is not h y d r o lyzed by sucrase or l e v a n s u c r a s e , is the specific substrate g e n e r a l l y used for assaying levanase act i v i t y in c r u d e extracts or culture supernatants. Since sucrose is h y d r o l y z e d by all t h r e e e n z y m e s and levans are h y d r o l y z e d s l o w l y by l e v a n s u c r a s e [61, these less specific substrates can only he used for assaying levanase a c t i v i t y in extracts or supernatants d e v o i d of sucrase a n d l e v a n s u c r a s e activities. Assays ~vere c a r r i e d out at 37°C in r e a c t i o n m i x t u r e s c o n t a i n i n g 0.2 M p o t a s s i u m p h o s p h a t e + 0.2 M p o t a s s i u m acetate buffer pH 5.5, samples

Leoanase synthesis by Bacillus subtilis mutants. of bacterial extracts [1] or culture s u p e r n a t a n t s dialyzed against the same buffer a n d either one of the three substrates : sucrose (240 mM), i n u l i n (10 raM), levan (1.3 raM). The r e a c t i o n Was stopped by h e a t i n g at 100°C for 5 min. and, if necessary, the p r e c i p i t a t e d p r o t e i n s were e l i m i n a t e d by centrifugation. Blanks of the same r e a c t i o n m i x t u r e s heated ~vithout p r e v i o u s i n c u b a t i o n at 37°C have always been included. Samples have been taken to d e t e r m i n e levanase activity w i t h the m e t h o d of Klotzsch and Bergmeyer [7] : the fructose (a) liberated after h y d r o l y s i s of these substrates has been c o n v e r t e d e n z y m a t i c a l l y to glucose-6-phosphate a n d d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y with tile use of glucose-6-phosphate dehydrogenase. One u n i t (U) of levanase activity is defined as tile a m o u n t of enzyme l i b e r a t i n g 1 ~tmol fructose

MAPPING

289

saeL MUTATIONS BY PBS1 T R A N S D U C -

OF

TION.

The sacL m a r k e r s have been m a p p e d i n a cluster b e t w e e n tile aroD and p h e A reference m a r k e r s by two factor t r a n s d u c t i o n crosses (table II, figure 1). sacL is also l i n k e d to leuA (17 per cent cotransfer) as was s h o w n by t r a n s d u c t i o n of strain QBIO1 (melC3 leuA8 trpC2) w i t h a lysate of QB166 (sacL5 lrpC2). PROPERTIES

OF LEVANASE SYNTHETIZED BY

sacL

MUTANTS.

Some properties of levanase have been s t u d i e d u s i n g crude extracts or culture s u p e r n a t a n t s of sacL m u t a n t s g r o w n in liquid m e d i u m with glutamate a n d succinate as c a r b o n sources. These

TABLE II. Tu,o factor transduction crosses involving sacL, pheA and aroD Markers. Recipient geootype pheA! trpC2 {6SY225) Recipient genotype aroDl~20 trpC?. ISBt20) Donor marker

sacL5 sacL6 sacL7 sacL8 sacL12

Selection

Phe +

Phe+ sacL Phe+

Per cent recombination

1101288 98•2OO 1011175 60/125 881175

62 51 57 52 50

Selection Aro +

Aro+ sacL Aro+

Per cent recombination

ND (c) 851290 1101294 32/170 441227

ND (e) 71 63 81 81

Conditions of transduction crosses are described in Materials and Methods. (c) Not determined.

per rain. or h y d r o l y z i n g 1 umol sucrose per nlin. u n d e r these c o n d i t i o n s . QUANTITATIVE SYNTHESIS.

DETERMINATION

OF

LEVANASE

Cultures g r o w i n g e x p o n e n t i a l l y at 37°C have been used to d e t e r m i n e i n t r a c e l l u l a r a n d extracellular specific activities of levanase w h i c h have been defined as i n t r a c e l l u l a r a n d extracel]ular activities per mg of p r o t e i n s y n t h e t i z e d w i t h i n the c u l t u r e Ecf. 8]. RESULTS. Mutations of Bacillus subtilis l e a d i n g to constitutive levanase synthesis have been o b t a i n e d as described in Materials a n d Methods. They b e l o n g p r o b a b l y to a single class, sacL, a c c o r d i n g to t r a n s d u c t i o n m a p p i n g results. (a) The fructose and glucose liberated after sucrose hydrolysis.

BIOCHIMIE, 1977, 59, n ° 3.

p r e p a r a t i o n s are essentially free from the other s a c c h a r o l y t i c enzymes, sucrase a n d ievansucrase, since these two enzymes are only synthetized in the p r e s e n c e of sucrose. Indeed, c h r o m a t o g r a p h i c analysis has s h o w n that levanase only is p r e s e n t i n these culture s u p e r n a t a n t s a n d cell-extracts.

- - chromatography. Levanase adsorbed on a h y d r o x y a p a t i t e c o l u m n was eluted w i t h 0.4 M potassium phosphate buffer pH 6.0. Sucrase a n d l e v a n s u c r a s e are eluted by 0.2 M a n d 1.4 to 2 M respectively. Thus the three enzymes can be eluted separately.

-- pH optimum. Optimal levanase activity has been o b t a i n e d at pH 5.5 i n 0.2 M phosphate acetate buffer.

- - catalytic properties. Levanase h y d r o l y z e s ~-1,2 a n d ~-2,6-fructofuranoside linkages. At pH 5.5, the a p p a r e n t K u values

290

F. Kunst

of levanase catalyzed h y d r o l y s i s of sucrose, i n u l i n (~-l,2-fructan) a n d levan (p~-2,,6-fructan) are respectively : 50 raM, 5 mM a n d 0.25 raM. The latter two values are calculated on a m o l e c u l a r weight basis of 5,000 for i n u l i n a n d 150.00 for levans. The rates of h y d r o l y s i s of these three substrates are very s i m i l a r (table III). --

stability.

Levanase can be stored at - - 2 5 ° C for several weeks w i t h o u t appreciable loss of activity. --

iuhibitors.

E n z y m a t i c activity is completely i n h i b i t e d b y a d d i t i o n of m e r c u r i a l s such as p - h y d r o x y m e r c u r i -

and coil. LEVANASE

SYNTHESIS.

Levanase is s y n t h e t i z e d a n d largely excreted into the culture m e d i u m b y s a c L m u t a n t s d u r i n g e x p o n e n t i a l growth i n l i q u i d C m e d i u m supplem e n t e d w i t h potassium glutamate a n d s u c c i n a t e as c a r b o n sources. However, levanase synthesis b y s a c L m u t a n t s is below the detection limit d u r i n g growth in liquid C mediunl supplemented with r a p i d l y metabolizable c a r b o n sources like sucrose, glucose or glycerol (table IV). This explains w i t h our previous failure to detect levanase activity on MM-glycerol plates. F r o m these observations we c o n c l u d e d that levanase synthesis b y s a c L mutants is not i n d u c i b l e b y sucrose a n d that this enzyme is subject to strong catabolite repression.

tP O

%

cj 4 thr

sacQ

arg C

J

:3 s4 :i! !

74

[

leuA

pheA

sacL

aroD

FIG. 1. - - The c h r o m o s o m a l m a p of B. subtilis Marburg according to H a r f o r d [9] and L e p e s a n t - I t e j z l a r o v a et al. [10]. The chromosomal segment indicates the position of the sacL magkers.

benzoate a n d m e r c u r i c chloride to final c o n c e n t r a tions of 10 -a M. BIOCHIMIE, 1977, 59, n ° 3.

s a c L is the only class of m u t a n t s i n w h i c h levanase synthesis could be detected : reference s t r a i n

Levanase synthesis by Bacillus 168 a n d t h e sacR c, sacSC a n d sacT c m u t a n t s l i s t e d i n t a b l e I d o n o t s y n t h e t i z e l e v a n a s e lo a d e t e c t a -

subtilis mutants.

291

s y n t h e t i z e l e v a n a s e at t h e s a m e r a t e as c o m p a r e d to t h e sacL r e f e r e n c e s t r a i n (QB2021).

TABLE III.

Levanase s y n t h e s i s by s a c L mulants. Specific activity (U/mg protein) Genotype

Strain

sacL5 sacL6 sacL7 sacL8 sacL12

QB166 QB167 QB168 QB169 QB171

lrpC2 lrpC2 IrpC2 lrpC2 trpC2

Intracellular

Extracellular

Sucrose

Levan

Inulin

Sucrose

Levan

Inulin

70 170 120 100 80

50 115 85 70 60

70 180 130 105 95

330 680 550 390 390

240 450 410 290 240

400 820 730 520 520

sacL m u t a n t s 'were gro'wn in C m e d i u m w i t h p o t a s s i u m g l u t a m a t e a n d succinate as c a r b o n sources. The specific activities of levanase 'were m e a s u r e d as the rates of h y d r o l y s i s of sucrose, i n u l i n or levans per mg of p r o t e i n s y n t h e t i z e d ~vith in the culture (cf. Materials a n d Methods).

ble level during cultivation in C medium mented with glutamate and succinate.

supple-

F u r t h e r m o r e , sacU 1~ m u t a t i o n s l e a d i n g lo o v e r p r o d u c t i o n of s e v e r a l o t h e r e x t r a c e l l u l a r e n z y m e s of Bacillus sublilis [111 d o n o t l e a d to o v e r p r o d u c -

TABLE IV.

I n f l u e n c e of d i f f e r e n t carbon sources on levanase synthesis.

Nature of carbon source

Specific activity of levanase (U/mg protein) Intracellular

Extracellular

180

820

Potassium glutamate

+

Potassium succinate Sucrose Glucose Glycerol

~5 <5 <5

lOO loo 100

DISCUSSION.

Bacillus subtilis 168 s y n t h e t i z e s t w o s a c c h a r o lyric enzymes after induction by sucrose : an intracellular sucrase and an extracellular levansuc r a s e . H o w e v e r , i t is s h o w n i n t h i s p a p e r t h a t another extracellular saccharolytic enzyme, called l e v a n a s e , is s y n t h e t i z e d b y m u t a n t s (sacL) d e r i v e d from this strain. A common r e g u l a t o r y g e n e (sacS) c o n t r o l s simultaneously sucrase and levansucrase synthesis [4, 5]. O n t h e c o n t r a r y , a s e p a r a t e c o n t r o l e x i s t s for levanase synthesis : 1. sacL m u t a n t s s y n t h e t i z e c o n s t i t u t i v e l y l e v a nase only. 2. c o n v e r s e l y , sacT, sacR a n d sacS m u t a n t s s y n thetize constitutively sucrase, levansucrase and both enzymes respectively, but do not synthetize levanase.

S t r a i n QB167 (sacL6 trpC2) was g r o w n i n C m e d i u m s u p p l e m e n t e d w i t h the indicated c a r b o n sources. Specific activities were d e t e r m i n e d as the r a t e s of i n u l i n h y d r o l y s i s per mg of p r o t e i n s y n t h e t i z e d w i t h i n the cultures as described in Materials a n d Methods. The detection l i m i t of i n t r a c e l l u l a r specific activity (5 U/rag) is l~wer t h a n the detection l i m i t of e x t r a cellular specific activity (100 U / m g ) since concentrated bacterial extracts h a v e been utilized in the first case.

L e v a n a s e s y n t h e s i s b y sacL m u t a n t s is r e p r e s s e d i n t h e p r e s e n c e of c a r b o n s o u r c e s l i k e g l u c o s e , glycerol or sucrose. However, if these mutants are grown in mineral medium supplemented only w i t h c a r b o n s o u r c e s of t h e t r i c a r b o x y l i c a c i d cycle (glutamate, succinate or citrate), release from catabo]ite repression occurs, which allows levanase synthesis.

t i o n of l e v a n a s e : c o n s t r u c t e d d o u b l e m u t a n t s of t h e g e n o t y p e sacL sacU ~ (QB20'19 a n d QB2020)

S u c r a s e a n d l e v a n s u c r a s e a r e i n v o l v e d i n suc r o s e c a t a b o l i s m [1, 41. S i n c e l e v a n a s e s y n t h e s i s is n o t s u c r o s e i n d u c i b l e t h i s s a c c h a r o l y t i c e n z y m e may have a different physiological function, for

BIOCHIMIE, 1977, 59, n ° 3.

292

F. Kunst

i n s t a n c e , d e g r a d a t i o n of l e v a n s a f t e r c a r b o n s o u r ce d e p l e t i o n . I n d e e d , l e v a n a s e a c t i v i t y h a s also b e e n d e t e c t e d i n w i l d t y p e B. s u b t i l i s s t r a i n s g r o w n o n l e v a n c o n t a i n i n g s o l i d m e d i u m [12] o r i n s u c r o s e g r o w n l i q u i d c u l t u r e s [13] ( R a p o p o r t , Jozon-Toulouse and Dedonder, unpublished res u l t s ) . I n t h e l a t t e r case, t h e a c t i v i t y h a s b e e n det e c t e d at t h e e n d of t h e e x p o n e n t i a l g r o w t h p h a s e when sucrose was mostly exhausted and ]evans were present at a high concentration. However, appropriate conditions for quantitative measurem e n t s of t h e r a t e of s y n t h e s i s of t h i s e n z y m e i n t h e s e s t r a i n s a n d i n s t r a i n 168 n e e d s t i l l t o b e defined. A eknolwledgme nls.

The generous gifts of b a c t e r i a l s t r a i n s f r o m C. Anagnostopoulos a n d N. H a r f o r d is g r a t e f u l l y acknowledged. W e w i s h to t h a n k our colleagues Rdgis C h a m b e r t , Genevi6ve Gonzy-Treboul, Georges R a p o p o r t a n d Martial Pascal for h e l p f u l suggestions a n d s t i m u l a t i n g discussions. W e are g r a t e f u l to Dani~le Lef6vre for the typing of the m a n u s c r i p t . R~SUM~. Une l~-D-fructofuranosidase - - appel~e l~vanase - qni h y d r o l y s e le saccharose, l ' i n u l i n e et les 16vanes a ~td identifi~es chez Bacillus subtilis Marburg. Cet e n z y m e n ' e s t pas d~celable d a n s la souche 168. Cependant, des m u t a t i o n s sacL - - localis~es sur le chromosome de la souche 168 e n t r e les m a r q u e u r s de r~f&-

BIOCH1MIE, 1977, 59, n ° 3.

a n d coll.

rence pheA et aroD - - c o n d u i s e n t h u n e synth6se constitutive de la lSvanase. Cette synth~se est rdprim6e p a r des sources de earbone telles que le glucose, le glyc6rol on le saecharose. REFERENCES. 1. Pascal, M., Kunst, F., Lepesant, J.-A. ~ Dedonder, R. (1971) Biochimie, 53, 1059-1066. 2. Pascal, M. & Dedonder, R. (1972) Carbohydr. Res., 24, 365-377. 3. Kunst, F., Pascal, M., Lepesant, J.-A., Walle, J. Dedonder, R. (1974) Eur. J. Biochem., 42, 611620. 4. Lepesant, J.-A., ~ u n s t , F., Lepesant-Kejzlarova, J. Dedonder, R. (1972) Molec. Gen. Genet., 118, 135-160. 5. Lepesant, J.-A., Kunst, F., Pascal, M., K e j z l a r o v a Lepesant, J., Steinmetz, M. ~ Dedonder, R. (1976) Microbiology, (American Society for Microbiology), pp. 58-69. 6. Rapoport, G. ~ Dedonder, R. (1963) Bull. Soc. Chim. Biol., 45, 493-513. 7. Klotzsch, M. ,& Bergmeyer, H. U. (1965) in <) (H. U. Bergmeyer ed.), 2nd p r i n t i n g revised, pp. 156-159, Academic Press, Nexv-York. 8. Kunst, F., Pascal, M., Lepesant-Kejzlarovh, J., Lepesant, J.-A., Billault, A..a Dcdonder, R. (1974) Biochimie, 5~, 1481-1489. 9. Harford, N. (1975) J. Bacteriol., 121, 835-847. 10. Lepesant-Kejzlarov~, J., Lepesant, J.-A., Walle, J., Billault, A. a Dedonder, R. (1975) J. Bacieriol., 1~1, 823-834. 11. Steinmetz, M., Kunst, F. a Dedonder, R. (1976) Molec. Gen. Genet., 148, 281-285. 12. Fuchs, A. (1959), Ph. D. Thesis, U n i v e r s i t y of Delft (The Netherlands). 13. Rapoport, G. (1966), Th~se de Doctorat d'Etat, Universit~ de P a r i s (France).