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,ersitdCiuude-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)
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. The
antibiotics for the presence droxy fatty acids, Iturinie antibiotics (bacihomycin D, bacilfomycin F, bacillomycin L, iturin and mycosubtilin) are antifungal peptidolipids synthesized by B. subtilis. They are characterized by the presence of @mine fatty acids that contain 14 to I7 carbon atoms but differ in the heptapeptidic 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 pamino fatty acids f5]. In this work, the relationship of compound X synthesis 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 Boulevard du 11 novembre 1918, F-69622 Villeurbanne Cedex, France.
of compound
X or @-hy-
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 antibiotics 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 analyzed 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-chromatography with standard ante&o C,,, iso C,j, II C,, P-hydroxy fatty acids. The iso C,, p-hydroxy fatty acid was identified by its retention time, using the linear relationships with the methyl esters of branched-chain [9].
2ooo I
0
1. Production
Chemicals Standard phospholipids were obtained from Sigma (U.S.A.); after TLC, they were revealed with molybdenum blue as described previously [4]. Standard P-hydroxy fatty acid methyl esters were obtained by hydrolysis 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, bacillomycin L, iturin and mycosubtilin) and two standard strains (B. subtilis 168 producing bacilysin, a noniturinic 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
40
30
into iturinic tration arbitrary
of bacillomycin
and time-course antibiotics
D excreted
unit corresponds
tion of iturin excreted corresponds incorporated
to 10
’
to IO-”
X.
iturin
pM.
Cm) or
(A 1 indicates
(0)
or bacillomycin
(A ) indicates medium. indicates
X isolated
ized with the producer of iturin or bacillomycin corresponds
[ “
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-hydroxy 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 surfactin previously described, five compounds which differ 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.
I
Determination
gf the location
Treatment
qf the
lactone
Amino
methylation _
reduction _
Asp
+
+ +
0.76
1.02 U.23
linkage
acid compositions GIu 1.23 0.83 O.lh
Leu 4.20 3.15 3.30
Val
1 1 1
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
%
ducing iturinic antibiotics, the influence of the culture medium on the nature of the carbon chains of P-hydroxy 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 producers 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 bacillomycin 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 maximum 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 concentration. 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 producing bacillomycin F grown in the presence o! valine
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 surfactin 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.
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 proFurther,
Od 0
0-
01 2
4
6
lsoleucine (g/l) Fig. 7. Effects of the addition X isolated from the producer of growth
8
0
2
4
6
Leucine (g/l)
8
0
2
4
6
8
Valine (g/l)
of branched a-amino acids (isoleucine, valine and leucine) on the nature of the fi-hydroxy fatty acids of compound of iturin (01, of bacillomycin D (A ), of bacillomycin F (0 1, of bacillomycin L ( A ), of mycosubtilin ( n ). After 100 h 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 spectrum 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.
TMS
r
PPM
Fig. 8. ‘H-NMR spectrum of the P-hydroxy fatty acid methyl esters of the compound X isolated the presence of valine at a 8 g/l concentration. As shown in Fig. 7 this analysis was performed
from the producer of bacillomycin on a major compound representing
F grown YO%.
in
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 phospholipids, 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. Phosphatidylethanolamine and phosphatidylglycerol were identified as labelled phospholipids. Their radioactivities were measured. For each strain, phosphatidylglycerol was about 15-fold more radioactive than phosphatidylethanolamine. Thus, the fatty acid methyl esters of phosphatidylglycerol were prepared and analyzed 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 producing bacillomycin D, bacillomycin F, bacillomycin L and iturin, respectively. By contrast, no radioactive /3-hydroxy fatty acids were dectected in the phosphatidylglycerol 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 phospholipids, unlabelled phosphatidylglycerol were prepared from the different strains and it was impossible to isolate P-hydroxy fatty acids from the phospholipids. Discussion Screening the B. subtilis strains producing bacillomycin 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 (Yamino acid composition of compound X is different
from that of iturinic antibiotics [l], compound X cannot be considered as a precursor of the iturinic antibiotics but it is interesting to notice that both compounds, iturinic antibiotics and compound X, are peptidolipids 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 presence 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 culture 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 phosphatidylglycerol, 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 phosphatidylglycerol is certainly a consequence of the synthesis of P-hydroxy fatty acids for surfactin. In conclusion, the B. subtilis strains which are characterized by the production of iturinic antibiotic synthesized 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 synthesis 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. Microbiol. 36. 164-16X.
5 Besson, F., Hourdou, M.L. and Michel, G. (I9YO) Biochim. Biophys. 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.
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,ersitdCiuude-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)
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. The
antibiotics for the presence droxy fatty acids, Iturinie antibiotics (bacihomycin D, bacilfomycin F, bacillomycin L, iturin and mycosubtilin) are antifungal peptidolipids synthesized by B. subtilis. They are characterized by the presence of @mine fatty acids that contain 14 to I7 carbon atoms but differ in the heptapeptidic 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 pamino fatty acids f5]. In this work, the relationship of compound X synthesis 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 Boulevard du 11 novembre 1918, F-69622 Villeurbanne Cedex, France.
of compound
X or @-hy-
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 antibiotics 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 analyzed 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-chromatography with standard ante&o C,,, iso C,j, II C,, P-hydroxy fatty acids. The iso C,, p-hydroxy fatty acid was identified by its retention time, using the linear relationships with the methyl esters of branched-chain [9].
2ooo I
0
1. Production
Chemicals Standard phospholipids were obtained from Sigma (U.S.A.); after TLC, they were revealed with molybdenum blue as described previously [4]. Standard P-hydroxy fatty acid methyl esters were obtained by hydrolysis 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, bacillomycin L, iturin and mycosubtilin) and two standard strains (B. subtilis 168 producing bacilysin, a noniturinic 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
40
30
into iturinic tration arbitrary
of bacillomycin
and time-course antibiotics
D excreted
unit corresponds
tion of iturin excreted corresponds incorporated
to 10
’
to IO-”
X.
iturin
pM.
Cm) or
(A 1 indicates
(0)
or bacillomycin
(A ) indicates medium. indicates
X isolated
ized with the producer of iturin or bacillomycin corresponds
[ “
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-hydroxy 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 surfactin previously described, five compounds which differ 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.
I
Determination
gf the location
Treatment
qf the
lactone
Amino
methylation _
reduction _
Asp
+
+ +
0.76
1.02 U.23
linkage
acid compositions GIu 1.23 0.83 O.lh
Leu 4.20 3.15 3.30
Val
1 1 1
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
%
ducing iturinic antibiotics, the influence of the culture medium on the nature of the carbon chains of P-hydroxy 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 producers 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 bacillomycin 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 maximum 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 concentration. 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 producing bacillomycin F grown in the presence o! valine
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 surfactin 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.
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 proFurther,
Od 0
0-
01 2
4
6
lsoleucine (g/l) Fig. 7. Effects of the addition X isolated from the producer of growth
8
0
2
4
6
Leucine (g/l)
8
0
2
4
6
8
Valine (g/l)
of branched a-amino acids (isoleucine, valine and leucine) on the nature of the fi-hydroxy fatty acids of compound of iturin (01, of bacillomycin D (A ), of bacillomycin F (0 1, of bacillomycin L ( A ), of mycosubtilin ( n ). After 100 h 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 spectrum 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.
TMS
r
PPM
Fig. 8. ‘H-NMR spectrum of the P-hydroxy fatty acid methyl esters of the compound X isolated the presence of valine at a 8 g/l concentration. As shown in Fig. 7 this analysis was performed
from the producer of bacillomycin on a major compound representing
F grown YO%.
in
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 phospholipids, 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. Phosphatidylethanolamine and phosphatidylglycerol were identified as labelled phospholipids. Their radioactivities were measured. For each strain, phosphatidylglycerol was about 15-fold more radioactive than phosphatidylethanolamine. Thus, the fatty acid methyl esters of phosphatidylglycerol were prepared and analyzed 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 producing bacillomycin D, bacillomycin F, bacillomycin L and iturin, respectively. By contrast, no radioactive /3-hydroxy fatty acids were dectected in the phosphatidylglycerol 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 phospholipids, unlabelled phosphatidylglycerol were prepared from the different strains and it was impossible to isolate P-hydroxy fatty acids from the phospholipids. Discussion Screening the B. subtilis strains producing bacillomycin 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 (Yamino acid composition of compound X is different
from that of iturinic antibiotics [l], compound X cannot be considered as a precursor of the iturinic antibiotics but it is interesting to notice that both compounds, iturinic antibiotics and compound X, are peptidolipids 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 presence 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 culture 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 phosphatidylglycerol, 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 phosphatidylglycerol is certainly a consequence of the synthesis of P-hydroxy fatty acids for surfactin. In conclusion, the B. subtilis strains which are characterized by the production of iturinic antibiotic synthesized 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 synthesis 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. Microbiol. 36. 164-16X.
5 Besson, F., Hourdou, M.L. and Michel, G. (I9YO) Biochim. Biophys. 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.
