© insrrrut PASTEUR/ELsEviEr Paris 1991

Res, Mierobiol. 199], 142, 757.764

Positive and negative regulation controlling expression of the sac genes in Bacillus subtilis M. D6barbouill~, I. Martin-Verstraete, M. Arnaud, A. Klier and G. R a p o p o r t ~,nitd de Biochimie Microbienne, URA 1300 du Centre National de la Re 'Terche Scientifique, Institut Pasteur, Ddpartement des Biotechnologies, 75724 Paris Cedex 15

Two saccharolytic enzymes, sucrose (sara gene product) and levansucrase (snob gene product), can be detected in crude extracts of Bacillus subtills after induction by sucrose, Sucrase is an intraeetiular enzyme, whereas levansucrase is secreted. Both enzymes are ~-D-fructofuranosidases, and levansucrase also catalyses the synthesis o f high molecular weight fructose polymers called [evan. Another gene, sacP, seems to form an operon with sacA. The sacP gene product is a membrane-bound component of the phosphotransferase system, Ell scr, specifically involved in sucrose uptake (Fouet el at., 1987). A third sucrose-hydrolysing enzyme called levanase (sacC gene product) is secreted. This [3-D-fructofuranosidase hydrolyses sucrose but also fructose polymers such as levan and inulin (Kunst et al., 1977). Sucrose and levanase from B. subtJlis share amino acid sequence similarities with yeast invertase (Fouetet at., L986; Martin et aL, 1987). However, no extensive se0uence similarities were found between members of this group and levansucrase. Specific ~nd pleiotropic regulatory mechanisms affe~ the expression of the sucrose system in B. subtilis (Lepesant et al., 1976). Recent results dealing with specific regulation within the sac system are presented in this review.

A) Positive

regulation

Most of the known regulatory gene products controlling the sucrose system are positive regulators. However, a special type of negative regulation which affects the activity of positive regulators has also been postulated (see section B).

The control o f sacPA and sacB gene expression The systematic isolation and analysis of mutants affected in saecharolytic enzyme synthesis has led to the characterization of regulatory genes controlling the sucrose system in B. subtilis (fig. 1). The saeB gear and the saePA operon are cantrolled by specific regulators: SacY and SacT, respectively (D~barbouill~ et aL, 1987; Aymerich and Steinmetz, 1987; Zukowski et al., 1990; Ddbarbouilld el aL, 1990). Both of these regulatory genes have been cloned and sequenced. Gene disruption experiments h ave showed that sac Y and sacT are positive regulatory genes. The deduced amino acid sequences of these two genes share strong similarity throughout their sequence, with 48 % of residues being identical. Moreover, these two proteins are homologous to BglG, the antiterminator of the [3-glucoside system (bgO in Escherichia cob (Steinmetz et aL, 1989; D6barbouill6 et M., 1990). Constitutive mutations previously located by genetic mapping in thesacS locus affect the sac Y gene and correspond to missense mutations (Crutz el aL, ].990). A single sacTeonstitutive mutant has been isolated. ~[he corresponding mutation has been sequenced and corresponds to a missense mutation, lmerestingly, constitutive mutations affecting the three antiterminators have been found in the same region (D~barbouill~ et al., 1990). These regulatory proteins SacT, SacY and BglG, are homologous proteins, which presumably function by similar mechanisms. Downstream from the sacB and saePA promoters are located two palindromic structures which resemble rho-independent transcription terminators (fig. 2). Deletion of the termination structure or sin-

M. DEBARBGUILLI~ E T AL.

758

gle base substitutions that destroy the dyad symmetry in the sucB palindromic structure led to constitutive synthesis o f levansucrase (Shimotsu and Henner, 1986; Steinmetz and Aymerich, 19g,6). S 1 mapping of thesacB promoter defined the transcription start site 199 bp upstream from thesacBgene, and it was shown that the region of dyad symmetry acts as a transcriptional terminator. Furthermore, the sacB promoter is constitutive and transcripts stop at the terminator in absence o f inducer. Transcripts extend past lhe terminator [n the presence of suciose (Shimotsu and Henaer, 1986). Comparison of DNA sequences around the palindromic sequences present downstream from the sacB and saePA promoters revealed significant homology. Fifty out of 53 bases are identical. The region of DNA sequence homology is also present in the []-glucoside utilization system of E. coil. The saeY and saeT genes probably encode antiterminator proteins required for the expression of sacB and sacPA, respectively. The strong similarity between the two regulatory systems raises the possibility of "cross-talk" between them. SacT is strongly similar to $aeY and their putative targets are also simitar. A residual induction of sncB expression was detected in a B. subtitis Asa'cYstrain al~d this low level of induction [s probably due to the SacT protein (Steinmetz et aL, 1989). A reciprocal phenomenon was observed in a B+ subtilis asacTstrain: a low level of sacPA e~pressiou was induced by sucrose, suggesting the involvement of the SacY antiterminatot (l~barbouil]~ et al., 1990). This hypothesis is reinforced by the fact that both suoB and saePh are constitutive in a strain containing a SacY constitutive mutation. This result indicates that a modified SacY aotiterminator induces sacPA expression. Wright and coworkers ha,,; recemi3 ~e~onztrated that the purified BglG andterminator is an RNAbinding protein tha', recognizes a specific sequence which Overlaps the upstream part of the two terminators of the bgl system. Mutational analysis of this sequeuce indicates that the protein requires a specific secondary structure of mRNA. Therefore, it was proposed that B;IG prevents transcription terminadot. by bind!uS to the nascent mRNA and preventing formation of the termination structure (Houman et aL, 1990). The homology found at the amino acid

level between SueT, SacY and BglG and the DNA sequence conservation between their putative targets strongly suggest that the bgl and sac genes are controlled by a similar molecular mechanism of antiterre[nation.

Levanase expression is controlled by a NifA-/ike regulator in B. subtilis sacC, which codes for levanase, the third saccharolytlc enzyme of 8. subtilia, belongs to a corn-

st__~.~sac Y •

l--'a~-q

=

I~tn!cr~se i

s~cT

i

)

pr~olcrreg~,

F"saeP

I

~,cA

1

s u ~ Lcan~poft ~ i ~ protein (Enzyme

nset)

Fig. i. Specificand pleiotropJc comrol mechanismsaffect. ing the expressionof the saePA and sacB gene ia.g. st¢blili~.

GGGA T TGTG;:C TGGTAJ%AGCAG GC AAGAC ~ TAA~A T FGCGTA~ATGbAAAAGGATCC-CTCTG~CC TTTATTCGTTGG CGAATTTTAGGTCT TT TTT

SaCF

GGG TTTGTTACTGATAAA GC ACSCAAGACC'fAAAA

saga

TGTAAAGGGCAAA~- - G T A ~ T T

TGGeG~CACttCT~ - ACATATTT?AGGTCT TT YTT

Fig. 2. Comparison of DNA sequences unstream of sucP, snob and bglF genes. A region of sirens similarity is boxed. This region corresponds to the BglG binding site observed in the mRNA Qf t]~. bgl opcron in E. coll.

R E G U L A T I O N O F sac G E N E E X P R E S S I O N

759

plex operon encoding a fructose-specific PTS. This

the levanasc operon is probably not controlled by an

gene is the distal gene of an operon containing four other genes called levD, levE, levF and levG (MartinVerstraete el aL, 1990). The expression of the B. subfilis levanase operon is inducible by fructose and subject to eatabolite repression (fig. 3). A fructose-inducible promoter has been eharacterr.zed by the primer extension method 2.7 kb upstream from the, ucC gene (Martin et aL, 1989). The upstream region of the Ievanase operon contains a positive regulatory gene called levR which is involved in the expression of the operon. A eonstitmive mutation (sacL8) was identified in levR. This mutation corresponds to a C~T transigon transforming a CAG codon (Gin 798) into TAG (stop) and eliminates 140 residues from the original polypeptide. Two lines of evidence strongly suggest that levR is a positive regulatory gene. 1) The sacL8 constitutive alle~e of levR, placed on a high copy plasmid, is dominant over the wild type chromosomal levR + allele; 2) disruption of t h e / e r r gene by introduction into the chromosome of a cassette containing a kanamyein resistance gene results in a total loss of inducibility of the levanase operon (fig. 3). The levR gene encodes a large polypeptide of 938 residues with a molecular m ~ s of 106 kDa. Two domains. A and B, containing respectively 2(10and [61 residues, were identified in LevR. The homology found between domain B and SacT, SacY and BglG is surprising, since

antitermination mechanism (fig. 4). Indeed, no transcriptional terminator with dyad symmetry was found between the promoter and the end of the eperon. The domain A of LevR shares similarity with the well conserved central domain of the NifA/NtrC family of Gram- activators (fig. 4). This central domain of NifA/NtrC is specifically required for the formation of open complexes between a54, the holoenzyme of RNA polymerase and the promoter. ATP and a specific activator are necessary to cataiyse formation of the corrcspondir.~; open promoters. An ATPbinding site is alst; present in LevR (fig. 4-). In Gram- bacteria, Ni?A and NtrC interact with specific upstream activating ~equences (UAS) and stimulate a class of promoters called - 1 2 / - 2 4 promoters. The consensus of these promoters is well established and differs from that of the - 1 0 / - 3 5 vegetative promoters, DNA sequence of the levanase promoter revealed at - t 2 / - 2 4 position~ two sequences it;.-~::icalwith those found in most NifA- and o54-controhed promoters. Eleven bases out of twelve are identical with the consensus of the 12/ 24 promoters (D~barbouill6 et at., 1991). Moreover, a putative UAS element (TGT-NI0-ACA) centered at position - 132 was also found upstream from the transcription start s.:te of the levanase opermL Deletion mapping experiments were done using ievDJacZ fusions. These constructions were reintroduced as

PJ,I~, pot ]/twe=rase'~ sigma54 J / levR ~ ~ ........... (( ..................... P • . ~ [ pos~ive I 'n presence regulator I ° f f r u ¢ l ° s e / / / f ~ P16

levlE I

sacS

IovF H

t"~/////////////////~-

H

I Plg

P28

P30

levanase

Jr:'

~y

in ab,~nce

" A-P

of fructose transport of fructose (P]S)

hydrolysis of sucrose levan and inulin

Fig. 3, Model of regulation of the levanase operon. The genetic organization of the tevanase operon of B_ xubrilis is represented. P represents the fructose-inducible promoter. The ievD, tevE, levFand levG gene products correspond to a fructosespecific PTS. tevR ¢ucodes a positive regulator. The activator may exist in 2 forms. (A-P), an inactive phosphorylated form or {A), an active ncu-phosphory~aLcd form.

M. D E B A R B O U I L L E E T A L .

760

single copies imo the chromosomal DNA of B. subtihs, Deletions encompassing this TGT-Nto-ACA sequence reduced the transcription rate more than ten-fold (1, Mariin-Verstraete et ul., unpublished results).

No gone encoding ~54 has yet been identified in B, subtilis, Using an E. colt ntrA mutant (~54-defectlve), it wa~ shown that l~vanase oparon expression in E. colt was strictly dependent on the presence of both levR free1 B. subtilis and n54 from E. coIL These results eonstitute the firs~ indication that n54 may be present in Gram ' bacteria (D~barhouilM el aL, 19911.

B) Involvement of the PTS in negative regulation of ~he sac g e n e s The phosphoenolpyruvate-carbohydrate phosphotransferase system is responsible for the uptake and concomitant phosphorylation of a number of sugars in both Gram + and G r a m - bacteria (Postma and LcUgClcr, 1985), During uptake by the PTS, a phosphoryl group is transferred from phosphocnolpyruvate to the different carbohydrates via a number of proteins which are transiently phosphorylated. Enzyme [ of the PTS and histidine contai.ning phosphocarrier protein (Hpr), which are cytoplasmic

A XifA N~fA

Kp H t E C

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~p n~zn

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niEA Ep H ~ / C

164 V ~ ' ~ S Q T ~ V X R A L n R B a P R

..... ~ A L ~ - - ~ K D ~ Z E ~ E ~ G ~

212

286

E~A~R~RK~F~L~D~T~L~GEa~ASF~AK~LRZL~E~ENE~GD~

340 ~28 268

25~

341 T L ~ - ~ Z ~ H L I E I V R L G H F R E D L Y Y ~ L N V ~ A L ~ Q E D Z A ~ L ~ 329 TLKV-D L KDLRN~/QNGEFREDLYY ZSGVP~Z~R~DGDI¥~LAR

RE~Tg~LF~I~TEHPSS~LLXT

~05 394 383

305 395 ~84 324

T~G~g~lKn~sv~nv~N~zng~r~o~xsn H~VR~I~HS~GRT~ISDGAIRL~MR~eWP~RE~ENC &~RFNE~-GRD~FAPSALD~nKCKFE~RI~ENC HW~Z&A~GV/&X~ETEM~TRL~R~NT

~45 4~4 ~22 363

2?3 213

E~T~I&~RV~RF~a~T=LL~GRI~P~E~LRV~E~r~V~GTK E~TVR~F~D~T~L~GDM~LDV~TR~LRVLAD~F~GYA ......

~L~'~RZP~T~Z~s~n~s~RR~VD~T

B n~ n8 ns n¢

LoyR SlUT $au~ BglG

ii .... B~IG

~K~RI~RHHAIV~-~EE ~Z ~ A ~ [ ~ [ A r N K K K ~ [ D ~ D P SK ~ - E ~ I •~n~TXIY~RN~V~D~E~V~J~GR~[~GF~KRA~ER~eSG~-E~LSSKE

RKDT P

e4 n l l ~ r l ~ z _ ~ l ~ m z ~ z a ~ ! ~ z ~ z n n K z n v x ~ n ~ a r ~ z ~ a ~ 5'7 L N C R L S E ~ f f ~ H ~ L ~ A T C D R ~ Z S ~ E R ~ G - ~ L Q D a Z Y Z S ~ D ~ C Q P A ~ P Q ~ N 109 L ~ Z L ~ H K ~ L ~ K ~ I ~ Z ~ W A Z G H ~ K ~ L g~g ~ p ~ n ~AG ~ A ~ n ~ - M D 111 n ~ N P L L S ~ L Y p E ~ W A~AL ~ K LG~O~G N~A~R ~A~R N 110 V ~ P M P L L N ~ RLYP~O~EEALT I~ D K R L G ~ I ~ D ~ G ~ A ~ H ~ V ~ M-

Fig. 4. Comparison of B. snbtifis LevR with siml]ar regulatory proteins. tA) Comparison of B. subrHis LevR with K. pneumoniae NifA and K+ pneumoniae NtrC. The ~aiao acid sequence of the A domains of four polypeptides are ~vcn in one.letter code and have been aligned by introducing gaps (hyphens) to maximize k:~ntity. Identical residues are boxed and combers indicate the position of the residues in the resl~ tire protein. LevR is 33 "70 identical in this domain with NifA el K. pneamoniae and R. meliloth ~,nd NtrC of K. pneumoniae, (B) Comparison of B. snbtilis LevR with B. subdlisSacT and Sac"/and E. coli 8glG. Similar resid.ue.sare boxed (accepted conservative replacements are I, L, V and M ; D and E ; A and G; R and K ; S and T: F and ¥). The percentages of similarhy between LevR and SooT, $aeY and BglG, in ~hese domain~, are 40, 42 and 34 o/0, respectively.

§3 5~

x~.o log

le3

165 163

R E G U L A T I O N O F sac OENE EXPRESSION proteins, are required for the transport and phosphorylation o f all PTS sugars and are therefore called general proteins. Sugar specificity in each uptake system is determined by specific proteins: a membrane-bound enzyme I1 and, in some ~:a~*,s, a.a associated soluble enzyme I[[. The PTS is aho implicated in several regulation mechanisms such as chemotaxis and transcriptional regulation. The involvement of a specific component of the P T s in indaction has been shown for the bgl opernn in E. edit (Mahadevan and Wrlght,' 1987), These authors pro-Bsl posed a model of regulation: the enzyme I1 , which is involved in [3-glucoside transport, exerts its negative regulation effect by phosphorylatiag the positive regulator BglG and thereby abolishing its activity (Amster-Choder et al., 1989; Schnetz and Rak, 1990). Control o f SaeY and SaeT activity Previous reports strongly suggest that specific components of the PTS also negatively regulate the expression of sac genes (Gay, 1979). Genetic evidence and DNA sequence analysis have shown that the sacs locus of B. subtilis contains two genes: sacX and sacY, The sacX gene codes for a protein of 459 amino acids sharing 56 % identity with the product of sacP, the sucrose-specific enz3ane ii scr and with variota enzyme II ~ from S, rautans, £. coti and Y. alginolvticus (Blatch el al., 1990; Zukowski et al., 1990). Since sac,° mutants do not transport sucrose, the sacX gene product is probably an inefficient Ell s~ or is poorly expressed (Fouet et al.. 19g9; Crutz el aL, 1990). The saeX gene o f B. s~Otilfs was detected upstream from sacY by mutagenesis using Tn917 tramposition. B, subtili$ strains containing such a iransposon showed constitutive sacB expression (Aymerich and Steinmetz, 198"/). It was also shown that an intenaal deletion of sacX resulted in u low constitutive sacB expression, suggesting that the saeXgeue product exerts its effect via a negative control ot the SacY antiterminator by phosphor~latiOlt as described for the bg( system. Moreover, this low constitutivity of snob observed in the Asac.X mutat.~t is ovetindueible by sucrose. These two phenoty[',es of the AsaeX strain are difficult to explain. It wa~ therefore suggested that in the absence of SacX, another regulator could partially inhibit SacY (Crutz et al., 1990). The sacPA operon is constitutively expressed at a low level in a B. subtilis ptsl mutant, in a strain deleted for ptsH, ptsl and ptsG, the expression of the operou is fully constitutive, thus confirming the involvement of the PTS in the induction process (D~barbouill6 et al.. 1990). The expression of the saePA opeton remains inducible in a A,sacX ~sacY strain. The sacX gene product is therefore not required for the control of the SacT atttitermioator. Moreover,

761

in a strain deleted for ptsh', ntsL prs:3 anu deleted for saeT, the saePA opeton expression is fully constitutive. In the same Apts background, when sacY is deleted, the sacPA aperon is not expressed (M, Arnaud, unpublished data). These results indicate that the activity of the two anliterminators is I~robably not regulated in the same way. SacY is thought to be active in the absence of phosphorylation, while SacT could require the pr~ence of an internal inducer transported by the PTS. Negative regulation o f the LevR activator by the fruetose-PTS The first four genes of the levanase operon levD, levE, levF attd levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of E. coil The levD and levg gene products are homologous to the N- and C-terminal part of ,he enzyme l i t " ' , respectively, whereas the levF and levG~enes products have similarities with the enzyme II ma". Surprisingly, the polypeptides encoded by the levD, 1evE, levFand levG genes are not involved in mannose uptake but ~orm a i'ructose phosphotransferase system in B. subtifis (MartinVerstruete et al., 1990). Constitutive fructose uptake was observed in a strain cop.raining a constitutive alIele of 1erR. As expected, the constitutive fructose uptake observed in the mutant is dependent on the general proteins of the PTS, enzyme 1 and Hpr. This uptake is abolished in a B. s.~btilis strain containing a ptsI mutation (Martin-Verstracte et al,, 1994)), Three constitutive mutants were previously isolated (Kunst et al., 1977) and the corresponding mutations sacL5, sacL6 and sacL7 were mapped and sequenced (table I). The seeL5 and saeL7 mutations were both located in the levE gene. The sacL5 mutation corresponds to a C to T transition in the 126th codon of lev2~, replacing Gln-126 by a stop codon. The sacL7 mutation is a G to A transition leading to the replacement of Trp,22 by a stop eodon, In strains carrying the sacL5 or the sacL 7 mutations, the presence of stop codons led to the synthesis of truncated LevE polypeptides. As those two mutants constitutively synthesized levanase, it was concluded that neither mutation has a polar effect on sacC gene expression, but that the levE polypeptide acts as a negative regulator of the levanase opeton. On the other hand, the aacL6 mutation is u G to A transition in the ievD geae. This mutation changes Gly-69 to Glu. In a merodiploid strain carrying the wiid type allele of levD ar.d the sacL6 allele. the expression of the levanase operon was indacible as in the wild type strain. These results suggest that levO acts as a repressor. Therefore, the levD and levE sen-~ p~oducts which are involved in a fructose-PTS uptake act as negative regulators of levanase operon expression. In addition, the inacti-

M. DEBARBOUILL[~ E T AL.

762

C

case of the levanase operon, two euzyme-I It-like are negative regulators rather than a composite enzyme 11. Moreover, LevR probably stimulates transcription by interacting with UAS and ~54 RNA polymeras¢, as observed for NifA and NtrC in Gram bacteria. This type of positive regulator differs from the antiterminators BglG, SacY and SueT.

C

Conclusion

Table I, Mutations affecting fructose uptake and expression of the levanase operon. Mutations

sacL6 mutation missense in levD G l y S ~ G l u s9 sacL7 mutation nonsense in /eve TroZ2~stop 22 sacL5 mutation nonsense in levE Glnt26~stop l~s sacL8 mutation nonsense in levR Gln79~ stop TM ptsl6 mutation Insertion of lacZ and erm genes in levG

Fructose uptake

ND

Expression of the levanase operon

C +

C C NI

ration of the levG geue led to the absence of expression of the levanase operon even in the presence of fructose. In a ptsl m-;tant, a constitutive expression of the levanase operon was observed (table I), The following model has therefore been proposed : in the presence of fructose, the polypeptides levD, levE, levF and levG with the general proteins of the PTS are involved in a phosphotransfer cascade leading to the transport and the phosphorylation of fructose. In the absence of inducer, the phosphate group is transterred to the LevR activator in the domain B, via the LevD and LevE polypeptides which are homologous to enzyme Ill of the mannose PTS. The interruption of the phosphotransfer cascade at the level of enzyme [, levD or levE polypeptides, prevents the inactivation of LevR leading to constitutive expression of the levanase operon (table I). However, LevF and probably LevG seem not to be involved in the phosphorylation of the activator. When LevG is not functional, the levanase operon is not expressed, even in the presence of fructose. The consequence of this inactivation which prevents fructose uptake, may lead to the permanent phosphorytation of the activator by LevD and LevE (Martin-Verstraete et al., 1990) (fig. 5). Although the bgl and levanase operons are both negatively controllcd by the PT$, their regulation differs in some ways. The enzyme I I age is composed of an N-termiual integral membrane domain and a C-terminal enzyme-llldike domain. In the absence of ILglucoside (inducer) the phosphate group is transferred from the enzyme-Ill-like domain to the antiterminator BglG (Schnetz and Rak, 1990). In the

The three enzymes saeA, sacB, and sac(2 are subject to a complex regulatory network involvingspecific activation in the presence of inducer (sucrose or fructose) and are also under a pleiotropic control. Two pleiotropic regulatory systems control the expression o f the sucrose operon, i) Catabolite repression: sncA and sacC gone expression is decreased in the presence of a carbon source such as glucose. Very little is known concerning the molecular mechanism of the glucose effect on these two genes, it) A second pleiotropic system involving the degS/degU, degQ and d e e r genes affects the expression of sacB. The dogs and clegU genes encode a "two-component" system (Henner et at., 1988 ; Kunst et aL, 1988 ; Tanaka and Kawata, 1988). As proposed for such a regulatory pair, the Dogs protein which perceives an as yet unidentified signal from the environment is autophosphorylated in the presence of ATP. In the wild type strain, DegS transfers its phosphoryl group to the of fetter DegU. In this phosphorylated form, the effector activates the expression of the target genes encoding degradative enzymes. In B. subtilis, a number of systems have recently been described in which protein phosphorylation is involved in processing sensory data and in activating gone expression. In the case o f two component systems (degS/degU, spolld/spoOA) the donor of phosphate is ATP (Amnniewski et al., t990; Dahl el at., 1991 ; Mukai et aL. 1990; Perego et al., 1989). The PTSmediated phosphorylation is dependent on the phosphoenolpyruvate and requires the presence of several proteins: a positiveregulator (BglG, SacY, SacT or LevR), the general enzymes of the P T S (enzyme I and probably Hpr) and. sugar-specific proteins (a membrane-bound cnzymc II) and in somc cases an associated enzyme Iil acts as a "sensor-like" component which responds to the presence of an eaterhal signal (inducer). This signal is transmitted throughout the PTS to the specific activators. In spite of tile functional similarity of these two types of regulatory pathways, there is no homology between the proteins involved in these types of regulation. Interestingly, in the sucrose system, phosphorylation of regulators by protein kinases or by the PTS is involved in specific and metabolic control and enables fine regulation of gene expression in response to environmental conditions.

REGULATION

O F sac G E N E E X P R E S S I O N

Aeknowledgemenls Work in the authors' laboralmy was supported by lie Centre National de la Recherche Sciernifique, Instimt Pasteur, Fondalion pour la Recherche M6dicale and tJnivgrfit.~ Paris VII. We thank Christine Ouga~t for t.~pcrt secretarial assislaacc and Jo~lle Bignon Ior excellent technical assistance.

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