51 Biochimica et Biophysics Acta, 1123 ( 1992) 5 l-58 0 1992 Elsevier Science Publishers B.V. All rights reserved 0005-276O/92/$05.00

BBALIP 53797

Synthesis of P-hydroxy fatty acids and /%-amino fatty acids by the strains of ~~~~~~1~~ ~~~~~~~~ producing iturinic antibiutics

Fraqoise Bessan *, Isabelle Tenoux, Marie-Laure Hourdou and Georges Michel

Laboratoire de Biochimie Microhienne CC. N.R.S. UMR 24), iJnic,ersitd Ciuude-Bemurd, Lyon (FrunceJ

(Received 22 Februa~ 1991t

(Revised manuscript received 13 August 1991)

Key words: fi-Hydroxy fatty acid; Surfactin; p-Amino fatty acid; Iturin; Bacikmycin; Mycosubtilin; (B. x&i/is)

The iturinic antibiotics, which contain long chain p-amino acids, are produced by ~~e~I~~s ~~~~~. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a Iipopeptide containing P-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar tn surfactin. The carbon chain of its p-hydroxy fatty acids was n C16, iso C16, iso C,, or ant&a C,,. The percentages of each &Lhydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-char a-amino acids to the culture medium_ These results demonstrate for the first time that iso C,, ~-hyd~~ fatty acid is a constituent present in such a surfaetin like iipopeptide. Besides, the presence of radioactive @-hydroxy fatty acids in the phospho~ipids when the strains were grown in the presence of sodium [‘*Clacetate seems also characterize the different strains producing iturinic antibiotics.

Iturinie antibiotics (bacihomycin D, bacilfomycin F, bacillomycin L, iturin and mycosubtilin) are antifungal peptidolipids synthesized by B. subtilis. They are char- acterized by the presence of @mine fatty acids that contain 14 to I7 carbon atoms but differ in the hep- tapeptidic region of the moIecufe [If. As such p-amino fatty acids has been described only in these antibiotics, it was interesting to study the biosynthesis of the iturinic antibiotics, Among them, the biosynthesis of iturin had been previously studied [2-41. Searching for putative fipopeptide precursors of iturin, a compound (X1 which contains /3-hydroxy fatty acids had been detected. The synthesis of this compound from acetate subunits seemed to be regulated by the presence of p-amino fatty acids. This had suggested a role of the P-hydroxy fatty acids as intermediates in the biosynthesis of p- amino fatty acids f5].

In this work, the relationship of compound X syn- thesis to iturinic antibiotic formation was examined by screening the different strains producing the iturinic

Correspondence: F. Besson, Laboratoire de Biochimie Microbienne (C.N.R.S. UMR 24), Universitd Claude-Bernard, Lyon I, 43 Boule- vard du 11 novembre 1918, F-69622 Villeurbanne Cedex, France.

antibiotics for the presence of compound X or @-hy- droxy fatty acids,

Materials and Methods

The origin of the strains of B. ~~~~~~i~ producing bacihomycin D, bacillomycin L, bacillomyci~ F, iturin and mycosubtilin has been previously described [l]. The standard strains were: B. subtilis SB 202 which does not produce iturinic antibiotic and was a gift from Dr. P. Hoet (ICP, Bruxefles, Belgium1 and B. ~~~~~~~~ 168 producing bacilysin, a non iturinic antibiotic [6,7f. These strains were grown at 35 0 C in a basal medium containing L-Glu and glucose [2] to which b,r..-Ile, Leu or Val was added.

Lipidic compounds were extracted with 5 mf of chloroform/methanol (2: 1) from the lyophilized 1 mI assays of incorporation of sodium [‘4Clacetate. In the case of the isolation of the lipidic compounds from cultures, 100 mg of IyophiIized cells were extracted with IO ml of chlorofo~/methanoI (2: 1). The antibi- otics and the compound X containing P-hydroxy fatty acids were isolated by column chromatography and preparative thin-layer chromatography (TLC) as previ-

52

ously described [8]. The fatty acid methyl esters were obtained by hydrolysis with 12 M HCl/methanol (30: 70) for 15 h at I10 o C; they were purified by preparative TLC on silica gel in hexanc/diethyl ether

(6 : 4).

Gas chromatography (GC)

The /?-hydroxy fatty acid methyl esters were ana- lyzed by GC on SP 2100 capillary column with an IGC

120 FL Intersmat apparatus. Temperature conditions were from 140 to 240 o C at a rate of 2 o C per min. The

/&hydroxy fatty acids were identified by co-chromatog- raphy with standard ante&o C,,, iso C,j, II C,, P-hy- droxy fatty acids. The iso C,, p-hydroxy fatty acid was

identified by its retention time, using the linear rela-

tionships with the methyl esters of branched-chain [9].

Spectrometric determination

The fast atom bombardment (FAB-MS) was made on a VG analytical ZAB-SEQ apparatus. The ‘H-NMR

spectrum was measured in carbon tetrachloride using a Briiker spectrometer at 300 MHz containing tetra-

methylsilane as internal standard.

Determination of radioactivity

The radioactivities of the different lipidic com- pounds were determined by liquid scintillation count- ing in a Tri Carb 2100 instrument (Packard Instru-

ments, Downers Grove, U.S.A.).

Chemicals Standard phospholipids were obtained from Sigma

(U.S.A.); after TLC, they were revealed with molybde- num blue as described previously [4]. Standard P-hy- droxy fatty acid methyl esters were obtained by hydrol- ysis of compounds containing well-identified P-hydroxy fatty acids (APD-II and APD-III, surfactin fractions

isolated by Dr. Hosono) [lo]; standard n C,,, n C ,(,

and II C,, ,&hydroxy fatty acids were kindly supplied by Dr. M. Bruneteau (UCB, Villeurbanne, France). After TLC, the P-hydroxy fatty acids were revealed by a rhodamine spray. Sodium [l-‘4Clacetate (1.85 GBq/mmol) was obtained from CEA (Saclay, France).

Results

Presence of compound X in the strains of B. subtilis producing iturinic antibiotics

The five B. subtilis strains producing the iturinic antibiotics (bacillomycin D, bacillomycin F, bacil- lomycin L, iturin and mycosubtilin) and two standard strains (B. subtilis 168 producing bacilysin, a non- iturinic antibiotic, and B. subtilis SB 202 which does not produce any antibiotic) were grown at 35 o C in the basal medium. At different time intervals, 1 ml of the different cultures were withdrawn and incubated for 1

2ooo I

0 IO 20 30 40

Time (h) Fig. 1. Production of bacillomycin D and iturin into the culture

medium and time-course of incorporation of sodium [ “<‘Iacetate

into iturinic antibiotics and compound X. (A 1 indicates the concen-

tration of bacillomycin D excreted into the culture medium. One

arbitrary unit corresponds to IO-” pM. (A ) indicates the concentra-

tion of iturin excreted into the culture medium. One arbitrary unit

corresponds to 10 ’ PM. Cm) or (0) indicates the radioactivity

incorporated into iturin or bacillomycin D, respectively. One arbi-

trary unit corresponds to one dpm. (0) or (0) indicates the radioac-

tivity incorporated into compound X isolated from the assays real-

ized with the producer of iturin or bacillomycin D. One arbitrary unit

corresponds to 20 dpm.

h at 35 o C with 0.1 MBq/ml of sodium [ “Clacetate and the assays were treated as described previously 151. The lipid extracts of the different assays were exam- ined by TLC in the solvent chloroform/methanol/ water (65: 25:4) and the radioactive compounds were detected by autoradiography. In the assays realized with the producers of bacillomycin F, bacillomycin L,

iturin and mycosubtilin, radioactive compound X was found. It co-migrated with the compound previously isolated [5]. By contrast, in the assays realized with the producer of bacillomycin D, compound X was absent. Fig. I compares the incorporation of sodium [l- ‘“Clacetate into the different compounds in the case of the producers of bacillomycin D and iturin. Both ra- dioactive bacillomycin D and iturin were synthesized when the assays were realized after IO-25 h of growth. This stage of growth, the begining of the stationary phase, corresponds to the excretion of each antibiotic into the culture medium. The maximum of radioactive compound X was synthesized by the strain producing iturin before 5 h of growth but it was not synthesized by the strain producing bacillomycin D, neither when radioactive antibiotic was present nor when it began to be excreted from the cells. With both strains which do not produce iturinic antibiotic, B. subtilis I68 or SB 202, no radioactive compound X was detected. Thus, it seems that the strains which do not produce iturinic antibiotic (even when it produces another antibiotic) are characterized by the absence of radioactive com-

53

600000 ,

bacillomycin D iturin bacillomycin F bacillomycin L mycosubtilin

Nature of the Bsubtilis producer

Fig. 2. Incorporation of sodium [‘“Clacetate into compound X and iturinic antibiotic. The radioactive precursor was added to a 50 ml culture of

the different strains, both compounds were prepared from the cells and their radioactivities were determined. (a) and (w) correspond,

respectively, to the radioactivities of compound X and iturinic antibiotic isolated from the five producers.

pound X. Besides, because compound X was absent in the strain producing bacillomycin D, it is impossible to claim that all the strains producing the iturinic antibi- otics synthesized compound X, as a possible intermedi-

ate of the antibiotics. As these assays were realized on 1 ml samples of the

culture and during only 1 h of incubation in the pres- ence of the radioactive precursor, the possible com- pound X synthesis was then studied by addition of

sodium [14Clacetate to the culture medium (18.5 MBq/mll at the time of inoculation of the B. subtilis

strains. At the onset of appearence of the antibiotic into the medium, the cells were harwested and total

lipids were extracted. The cellular extracts of the dif-

ferent strains were analyzed for the presence of ra- dioactive compound X. The cellular extracts of the five strains producing iturinic antibiotic contained radioac-

tive compound X and antibiotic whereas the non-pro- ducer strains did not. Fig. 2 compares the radioactivi- ties of compound X and antibiotic. It appears that, in these conditions, the producer of bacillomycin D syn- thesized radioactive compound X. The radioactivity of

compound X isolated from the B. subtilis strain pro- ducing bacillomycin D was similar to those of com- pound X isolated from the strains producing mycosub- tilin or bacillomycin L. Besides, whatever the B. subtilis

4000 3doo 2000 W;,4;NUMBER

(CMIJIO 900 800 700

54

strain producing iturinic antibiotic, compound X was always more radioactive than the antibiotic. In these assays realized on the cellular extract of the five pro- ducers, compound X was enough concentrated to be detected according to the Irmscher revelation [l l] which used the hydrophobic properties of the P-hy- droxy fatty acids of compound X.

Characterization of compound X In order to establish its nature and its relationship

to the biosynthesis of iturinic antibiotic, the structure of compound X was studied. Purification yielded a colorless powder with a melting point at 13%139”C, that gave a positive reaction with rhodamine but a negative one with ninhydrin and Pauly reagent. OnIy a single spot was observed by TLC on silica gel in chloro- form/methanol/water (65 : 25 : 4) (R, 0.71) or in chlo- roform/ methanol (5 : 11 (K, 0.15). The I.R. spectrum (Fig. 3) indicated the presence of peptide bonds at 1625 and 1520 cm-’ and an ester linkage at 1700 cm-‘. Quantitative analysis of the a-amino acids [12] gave the following ratios: Asp,,,, Glu,,,,, L~LI~,~~,, Val,,

With regards to the peptidolipids previously de- scribed, two compounds, esperin 1131 and surfactin 1141, both contain P-hydroxy fatty acids and the a-amino acids: Asp, Giu, Leu and Val in the same ratio (1 : 1 : 4: 1) and possess the same sequence: P-hydroxy fatty acids --j Glu --f Leu + Leu + Val -+ Asp --) Leu --+ Leu. They differ, however, by the position of the lactone linkage. Since the 1.R. spectrum of compound X indicated the presence of a lactone ring (Fig. 3), it was assumed that its formation invoIved the P-hydroxyl group of the P-hydroxy fatty acids and a carboxyl group of either Glu or Asp or the C-terminal a-amino acid residue of the peptide chain. The lactone was reduced with LiAlH4 [IS] either without any treatment or after methylation with diazomethane [8]. The compound was then hydrolyzed by 6 M HCl for 8 h at 130’ C and the number of a-amino acid residues was determined in comparison with the a-amino acid content of native compound X. The data are summarized in Table I. When compound X was reduced without methylation, only three of the four Leu residues were evident; however, when it was methylated before reduction, Glu, Asp and one Leu residue disappeared. From these results, it was concluded that lactone linkage

TABLE I

Determination gf the location qf the lactone linkage

Treatment Amino acid compositions

methylation reduction Asp GIu Leu Val _ _ 1.02 1.23 4.20 1 - + 0.76 0.83 3.15 1 + + U.23 O.lh 3.30 1

BHFA’ -> L-Glu -z L-Leu -> 0-Leu -> L-Vial -&Asp -> D-Leu -> L-Leu

I I

Fig. 4. Structure of surfactin. BHFA” = CJ-hydroxy fatty acids.

formation occurred between the carboxyl group of the C-terminal Leu and the hydroxyl group of the fi-hy- droxy fatty acids. As compound X co-migrated with standard surfactin (fractions APDII and APDIII) by means of TLC in different solvent systems, the lipopeptide seems to be very similar to surfactin (Fig. 4).

Besides, surfactin had been characterized by its haemolytic properties [16]. In order to know whether compound X has any activity, compound X was added at different concentrations on blood agar plates and Fig. 5 shows the haemolytic activity of compound X. At a 100 pg/ml concentration, compound X induces a zone of haemolysis. This result confirms the chemical similarities of compound X with surfactin.

Identification of the p-hydroxy fatty acids of compound X isolated from the different strains of B. subtilis

With regards to the ,&hydroxy fatty acids of sur- factin previously described, five compounds which dif- fer by the nature of their carbon chain have been identified; they are iso C,,, anteiso CT,,, n C,,, iso C,, and anteiso C,, P-hydroxy fatty acids [10,14]. Thus, compound X was recovered from cultures of the five antibiotic-producers, the P-hydroxy fatty acid methyl esters were prepared and identified by GC. Fig. 6 provides the results when the different strains were grown in the standard medium. For each strain, the major component was anteiso C,5 P-hydroxy fatty acids.

Fig. 5. Haemolytic activity of compound X assayed on Columbia

blood agar plates CbioMirieux, France). 200 pl of carbonate buffer 50 mM (pH 9.5) containing 0 (A), 70 (B) or 100 ~&/ml CC) of

compound X was used.

55

% 8oo 60

1

40

20

0 iC14 nC14 iC15 aiC15

Fig. 6. Nature of the carbon chains of the P-hydroxy fatty acids of

surfactin isolated from the strains producing the different iturinic

antibiotics. The P-hydroxy fatty acids were prepared from the sur-

factin synthesized by the producer (from left to right) of iturin (dark

close hatched bar), of bacillomycins D (close hatched bar), F (solid

bar), L (open bar), of mycosubtilin (hatched bar); they were analyzed

by GC. The results are expressed in percentage of total P-hydroxy

fatty acids. i = Iso; ai = ant&o.

Further, the B. subtilis producers of bacillomycin F and mycosubtilin contain 2-4% of n C,, P-hydroxy fatty acids and 21-33% of iso Ci4; by contrast, the B. subtilis producers of bacillomycin D, bacillomycin L and iturin contain 19-32% of 12 C,, P-hydroxy fatty acids and only O-8% of iso C,,. Thus, it is the first time that the presence of iso C,, P-hydroxy fatty acid in such a surfactin like compound has been described.

As C,, P-hydroxy fatty acids were not found in the compounds X isolated from the B. subtilis strains pro-

Od 01 0 2 4 6 8 0 2 4 6 8

lsoleucine (g/l) Leucine (g/l)

ducing iturinic antibiotics, the influence of the culture medium on the nature of the carbon chains of P-hy- droxy fatty acids was studied. The addition of Val, Lcu or Ile to the medium modified the composition of the /3-hydroxy fatty acids and Fig. 7 shows the effect of branched-chain a-amino acids on the percentages of the increased P-hydroxy fatty acids. With the five pro- ducers of antibiotics, the addition of Ile increased ante&o C,, P-hydroxy fatty acids and Leu iso C,, P-hydroxy fatty acids. The more prominent increases were observed with anteiso C 1s p-hydroxy fatty acids in the case of the B. subtilis producing mycosubtilin and bacillomycin F while they were iso C,, P-hydroxy fatty acids in the case of the B. subtilis producing bacil- lomycin D, bacillomycin L and iturin (Fig. 7). The addition of Val increased iso C,, /3-hydroxy fatty acid for the producers of bacillomycin F, bacillomycin L, mycosubtilin and iturin. As valine is known to be a precursor of the even iso carbon chains [ 171, these results confirm the previous identification of the iso C,, P-hydroxy fatty acid. In the case of the B. subtilis strain producing bacillomycin D, no iso C,, P-hydroxy fatty acid was found in the standard medium and the addition of Val did not modify this result (Fig. 7). On the other hand, the strain which produces the maxi- mum of iso C,, /3-hydroxy fatty acid (90%) was the B. subtilis strain producing bacillomycin F when it was grown in the presence of valine at a 8 g/l concentra- tion.

Further characterization of iso C,, /3-hydroxy fatty acid was made by mass and ‘H-NMR studies of the compound X isolated from the B. subtilis strain pro- ducing bacillomycin F grown in the presence o! valine

0- 0 2 4 6 8

Valine (g/l)

Fig. 7. Effects of the addition of branched a-amino acids (isoleucine, valine and leucine) on the nature of the fi-hydroxy fatty acids of compound

X isolated from the producer of iturin (01, of bacillomycin D (A ), of bacillomycin F (0 1, of bacillomycin L ( A ), of mycosubtilin ( n ). After 100 h

of growth at 35 o C, the /3-hydroxy fatty acid methyl esters of surfactin were prepared and analyzed by GC.

56

at a 8 g/l concentration and its /3-hydroxy fatty acid which represents 90% of the compound X isolated in such conditions.

On the one hand, the FAB-MS spectrum of the compound X displayed a major peak (M + H)+ at m/z

1023; this result agrees with a C,, P-hydroxy fatty acid and confirm the n-amino acid composition previously

determined for compound X. On the other hand, the

P-hydroxy fatty acid methyl esters displayed a major peak (M + HI+ at m/z 259; this result agrees with a C,, P-hydroxy fatty acid methyl ester.

The pattern of the proton magnetic resonance spec- trum of these P-hydroxy fatty acid methyl esters and

the assignment of the signals are given Fig. 8. A doublet signal corresponding to six hydrogen atoms was present at 0.85 ppm, showing the presence of a

R - (CH2)n - CHOH - CH2 - COOH

Fig. Y. Structure of the P-hydroxy fatty acids of compound X.

R = CH,-CH, and II = Y in the case of 12 C,, /!-hydroxy fatty acid:

R = CH,-CH(CH,) and II = X in the case of ho C,, ,&hydroxy

fatty acid: R = CH,-CH(CfI,) and II = Y in the case of Iso C‘,<

P-hydroxy fatty acid; R = CH,-CH2-CH(CHIl and 11 =X in the

case of ar~~cko C ,F /3-hydroxy fatty acid.

terminal isopropyl group. This spectrum is very similar to the spectra of iso C,, P-hydroxy fatty acid and z’so

C,, @-hydroxy fatty acid isolated from siolipin [18]. In conclusion, the mass and ‘H-NMR studies confirm the previous identification of the iso C,, /3-hydroxy fatty acid.

Fig. 9 shows the structure of the /?-hydroxy fatty acids of compound X.

r

TMS

PPM

Fig. 8. ‘H-NMR spectrum of the P-hydroxy fatty acid methyl esters of the compound X isolated from the producer of bacillomycin F grown in the presence of valine at a 8 g/l concentration. As shown in Fig. 7 this analysis was performed on a major compound representing YO%.

57

Presence of P-hydroxy fatty acids in the phospholipids of the strains producing iturinic antibiotics

As the different iturinic antibiotic producers are

characterized by their ability to synthesize compounds containing P-derivatives of fatty acids (P-hydrcxy fatty

acids or p-amino fatty acids), the nature of the carbon chains of other compounds containing fatty acids was then studied. When sodium [‘4C]acetate was added to

the culture medium of the different B. subtilis strains,

the most radioactive compounds present in the cellular extract are iturinic antibiotic, compound X and phos-

pholipids, the presence of which was determined by molybdenum blue reaction [4]. The phospholipids of the strains producing iturinic antibiotics was analyzed

in comparison with those of the standard strains. Phos- phatidylethanolamine and phosphatidylglycerol were identified as labelled phospholipids. Their radioactivi- ties were measured. For each strain, phosphatidyl-

glycerol was about 15-fold more radioactive than phos- phatidylethanolamine. Thus, the fatty acid methyl es- ters of phosphatidylglycerol were prepared and ana- lyzed by TLC. Labelled P-hydroxy fatty acids were identified in all the strains producing antibiotic; the

radioactive P-hydroxy fatty acids represent 19, 22, 20 and 15% of the radioactivities of total fatty acids of phosphatidylglycerol isolated from B. subtilis produc- ing bacillomycin D, bacillomycin F, bacillomycin L and iturin, respectively. By contrast, no radioactive /3-hy- droxy fatty acids were dectected in the phosphatidyl- glycerol isolated from the standard strains which do not produce antibiotic. If they were present, they must be inferior to 0.5%. In order to identify the nature of the carbon chains of the P-hydroxy fatty acids of phos- pholipids, unlabelled phosphatidylglycerol were pre- pared from the different strains and it was impossible to isolate P-hydroxy fatty acids from the phospholipids.

Discussion

Screening the B. subtilis strains producing bacil- lomycin D, bacillomycin F, bacillomycin L, iturin and mycosubtilin for the presence of possible precursors of the iturinic antibiotics, we isolated a compound X

containing /3-hydroxy fatty acids from all these strains. As this compound was absent in the two standard strains of B. subtifis which do not produce iturinic antibiotic, it was interesting to determine its structure. Compound X is a peptidolipid containing the a-amino acids: Asp, Glu, Leu, Val in the molar ratios 1 : 1: 4: 1. The position of the lactone linkage of this peptidolipid was studied. Even if the sequence of the a-amino acids was not studied, the determination of the chemical properties and haemolytic activity of compound X shows that compound X and surfactin (Fig. 4) are identical or structurally very similar. Because the (Y- amino acid composition of compound X is different

from that of iturinic antibiotics [l], compound X can-

not be considered as a precursor of the iturinic antibi-

otics but it is interesting to notice that both com-

pounds, iturinic antibiotics and compound X, are pep- tidolipids constituted by a P-derivative of fatty acids

(p-amino fatty acids and /3-hydroxy fatty acids) and seven a-amino acids residues. Nevertheless, the pres-

ence of compound X appears to be correlated with the

iturinic antibiotic synthesis by the B. subtilis strains as compound X is completely absent in the B. subtilis strain producing bacilysin and in the B. subtilis strain

which do not synthesize any antibiotic. As it seems logical that both /?-hydroxy fatty acids

and p-amino fatty acids have the same intermediates

for their carbon chain synthesis, it was interesting to determine the nature of the carbon chains of the P-hydroxy fatty acids of compound X isolated from the

strains producing iturinic antibiotic. In all the cases, the lipidic part of compound X was identified to n C,,, iso C,,, iso C,, and anteiso C,, /3-hydroxy fatty acids. The nature of the carbon chains of the P-hydroxy fatty

acids was independent of the composition of the cul- ture medium; only modifications of the percentages of

each /3-hydroxy fatty acid were observed when

branched-chain a-amino acids were added to the medium (Fig. 7). The nature of the carbon chains of

the P-hydroxy fatty acids of compound X (Fig. 6) can be compared with the nature of the carbon chains of

the P-amino acids. These differences correspond to those determined in this study with the P-hydroxy fatty acids of surfactin (Fig. 7).

In order to better characterize the relation between the biosynthesis of p-amino fatty acids and P-hydroxy fatty acids, the lipidic cell extracts obtained in the presence of sodium [“Clacetate were screened for the presence of compounds containing P-hydroxy fatty acids or p-amino fatty acids, different from surfactin or iturinic antibiotic. Radioactive /3-hydroxy fatty acids

were isolated from radioactive phosphatidylglycerol but, as they cannot be isolated from unlabelled phospha- tidylglycerol, P-hydroxy fatty acids do not represent a significant part of phosphatidylglycerol. Nevertheless, radioactive P-hydroxy fatty acids were decteted only in the B. subtilis strains producing iturinic antibiotics while they were absent in the two other strains. The presence of radioactive /3-hydroxy fatty acids in phos- phatidylglycerol is certainly a consequence of the syn- thesis of P-hydroxy fatty acids for surfactin.

In conclusion, the B. subtilis strains which are char-

acterized by the production of iturinic antibiotic syn- thesized another lipopeptide: surfactin. It is interesting to notice the complementary activities of these two compounds: iturinic antibiotics (bacillomycins D, F, L, iturin and mycosubtilin) possess antifungal properties [19-211 and surfactin antibacterial ones [22]. The syn- thesis of the carbon chains of their lipidic part, P-hy-

58

droxy fatty acids and p-amino fatty acids, seems to be regulated by the same factors.

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique UMR 24. We thank Dr. K. Hosono of Fermentation Research Institute (Ibaraki, Japan) for the gift of surfactin samples (i.e. APDII and APDIII). We are also indebted to M.C. Duclos of Laboratoire de Biochimie VCgCtale et Mycochimie (Villeurbanne, France) for the I.R. absorbance spectra. We are pleased to thank Dr. B. Fenet of Centre de RMN de l’Universit6 Claude Bernard (Villeurbanne, France) for NMR spectrometric analyses and his useful discussion.

References

Hourdou, M.L.. Besson, F.. Tenoux, 1. and Michel, G. (1989)

Lipids 24, 940-946.

Besson, F.. Chevanet. C. and Michel. G. (1987) J. Gen. Microbial.

133. 767-772.

Hourdou. M.L.. Besson, F. and Michel. G. (198X) J. Antibiotics

41, 207-21 I.

Hourdou. M.L.. Besson, F. and Michel, G. (1090) Can. J. Micro-

biol. 36. 164-16X.

5 Besson, F., Hourdou, M.L. and Michel, G. (I9YO) Biochim. Bio-

phys. Acta 1036, 107-106.

6 Besson. F., Peypoux, F.. Michel, G. and Delcambe. L. (197X) J.

Antibiotics 3 1, 2X4-288.

7 Walker, J.E. and Abraham, E.P. (1970) Biochem. J. 178. 503-570.

8 Besson, F. and Michel. G. (1088) J. Antibiotics 41. 2X2-28X.

Y Kawanami. J. (1977) Chem. Phys. Lipids 7. 150-177.

10 Hosono, K. and Suzuki, H. (19X3) J. Antibiotics 36. 667-673.

11 Irmscher. G.. Bovermann, G., Boheim, G. and Jung, G. (lY7X)

Biochim. Biophys. Acta 507. 470-4X4.

12 Ghuysen, J.M.. Tipper, D.J. and Strominger, J.L. (1966) Methods

Enzymol. X. 6X5-699.

13 Thomas. D.W. and Ito. T. (IYhY) Tetrahedron 25, IYXS-1900.

14 Hosono, K. and Suzuki, H. (19X3) J. Antibiotics 36, 674-67X.

15 Lewis, B.A., Smith, F. and Stephen, A.M. (1963) in Methods in

Carbohydrate Chemistry. Vol. 2 (Whistler, R.L. and Wolfram,

M.L.. eds.), pp. 6X-76. Academic Press, New York.

16 Nakano. M., Mahariel, M. and Zuber. P. (19xX) J. Bacterial. 171).

5662~5668.

17 Kaneda, T. (1977) Bactcriol. Rev. 47. 3Y7-47X.

18 Kawanami, J. and Otsuka, H. (1960) Chem. Phy\. Lipids 3.

13.5-139.

19 Besson, F., Peypoux, F.. Michel, G. and Delcambe, L. (1979) J.

Antibiotics 32. 136-140.

20 Mhammedi, A., Peypoux. F.. Besson, F. and Michel. G. (1082) J.

Antibiotics 35, 306-3 I 1. ?I Raubitschek, F. and Doxtrovsky. A. (ISSO) Dermatologica 100.

45-49.

22 Tsukayoshi, N.. Tamura, G. and Arima. K. (1970) Biochim.

Biophys. Acta 196. 204&270.