Characterization of Surfactin-like Cyclic Depsipeptides Synthesized by

Bacillus pumilus from Ascidian Halocynthia aurantium

Natalie I. Kalinovskaya,1* Tatyana A. Kuznetsova,1 Elena P. Ivanova,1** Ludmila A.

Romanenko,1 Valery G. Voinov,1 Felix Huth,2 and Hartmut Laatsch2

1Pacific Institute of Bio-organic Chemistry of the Far-Eastern Branch of the Russian

Academy of Sciences, 159 pr. 100-Let Vladivostoku, 690022 Vladivostok, Russian Federation 2Department of Organic Chemistry, University of Goettingen, Tammanstr. 2. D-37077

Goettingen. Germany

* Corresponding author; telephone: +7(423 2)311168; fax: +7(423 2)314050; e-mail:

piboc@stl.ru

** Present address: Swinburne University of Technology, 533-545 Burwood Rd, Hawthorn,

33122, Melbourne, Australia

Running title: Surfactin-like cyclic depsipeptides from Bacillus pumilus

Key words: Surfactin analog, cyclic depsipeptides, Bacillus pumilus, ascidian Halocynthia

aurantium

Summary: A marine bacterium (KMM 1364), identified as Bacillus pumilus, was isolated

from the surface of ascidian Halocynthia aurantium. Structural analysis revealed that the

strain KMM 1364 produced a mixture of lipopeptide surfactin analogs with major

components ranging in size from molecular masses of 1035, 1049, 1063 and 1077. The

variation in molecular weight represents changes in the number of methylene groups in the

lipid and/or peptide portion of the compounds. Structurally, these lipopeptides differ from

surfactin in the subsitution of the valine residue in position 4 by leucine, and have been

isolated as two C-terminal variants, with valine or isoleucine in position 7. As constituents of

the lipophilic part of the peptides, only β-hydroxy-C15-, β-hydroxy-C16- and a high amount of

β-hydroxy-C17 fatty acid were determined.

INTRODUCTION Biosurfactants comprise a structurally diverse group of surface-active compounds produced

by microorganisms. Increasing interest to these substances is explained by the wide range of

their biotechnological applications including enhanced oil recovery due to reduction of

surface and interfacial tensions in aqueous solution and hydrocarbon mixtures (Singer, 1985),

deemulsification (Cairns et al., 1982; Mulligan, and Gibbs, 1993), health care (Muller-

Hurting et al., 1993), and food-processing industries (Velikonja and Kosaric, 1993).

Microbial surfactants are of particular interest because they, in contrast to synthetic analogs,

are biodegradable and can be produced through fermentation on renewable substrates.

The biosurfactant-producing microorganisms belong to different taxa (Desai and Banat,

1997). However, among Gram-positive spore-forming bacteria only a few species of the

genus Bacillus (i.e. B. subtilis and B. licheniformis), Brevibacillus brevis (former Bacillus

brevis) and Paenibacillus polymyxa (former Bacillus polymyxa) are known producers of

cyclic lipopeptides including decapeptide antibiotics (gramicidins) and lipopeptides

antibiotics (polymyxins) with surface-active and chelating properties. The cyclic lipopeptide

surfactin from B. subtilis was intensively structurally studied in respect to its biotechnological

and pharmacological applications (Cooper et al., 1981; Vollenbroich et al., 1997). A surfactin

like lipopeptide surfactant, lichenisin, is produced by B. licheniformis JF-2 and was patented

as an enhanced oil recowery agent (McInerney et al., 1985). The structure, molecular

genetics, properties, and production of biosufactants have been reviewed recently (Desai and

Banat, 1997; Sullivan, 1998).

Over the last decade we studied the biologically active secondary metabolites

produced by marine bacteria isolated from both seawater and an array of marine invertebrates

(Elyakov et al., 1996). Previously we reported about cyclic depsipeptides found in B. pumilus

isolated from the sponge Ircinia sp. (Kalinovskaya et al., 1995). This paper describes the

isolation and structure elucidation of one of the isoforms of surfactin produced by B. pumilus,

isolated from surface of cuticula of the ascidian Halocynthia aurantium, common inhabitant

of the Sea of Japan.

MATERIALS AND METHODS

Instruments

UV spectra were recorded on a Specord UV-VIS M 40 spectrophotometer (Carl Zess Jena) in

methanol. IR spectra were obtained on a Specord M-82 (Carl Zeiss Jena) spectrometer in CHCl3.

The amino acid composition was determined on an amino acid analyzer (Biotronic LC 2000,

Sweden, using DS-6A resin column) after total hydrolysis of the lipopeptides in 5.6 N HCl at

105oC for 48 h. NMR spectra were recorded on a Bruker AM 500 spectrometer (500 and 125.7

MHz for 1H and 13C, respectively, using TMS as the internal reference). The native peptides

were analyzed by fast atom bombardment mass spectrometry (FABMS) on a Finnigan 95 A

mass spectrometer, using a 3-nitro-benzaldehyde matrix (3-NBA). Positive and negative ions

were detected.

Bacterial strain

The strain Bacillus pumilus KMM 1364 was isolated from the ascidian Halocynthia aurantium in

August 1989, the Troitza Bay, Gulf of Peter the Great (Romanenko et al., 2001). This strain was

grown on a rotary shaker (120 rpm) in 1 L Erlenmeyer flasks containing 200 ml of the medium

(g/L): K2HPO4 (0.07), NH4Cl (1.0 ), yeast extract (5.0 ), FeSO4 (0.025 ), 1 M Tris buffer (20 ml),

artificial sea water (200 ml), distilled water (800 ml), pH adjusted to 7.5, during 20 h at 24-26oC.

Isolation of Depsipeptides 1, 2, 6-8

The cells (from 6 L of culture medium) were collected by centrifugation at 5000 g for 40 min

and suspended in water (50 ml), frozen and destructed by ultrasonic treatment. The suspension

was extracted with a mixture of chloroform-methanol (3:1) and the organic phase evaporated to

dryness. Column chromatography of the сrude extract (402 mg) on a silica gel column (40/100

µ, Chemapol, Czechoslovakia, 1.5x20 cm) with solvents of increasing polarity gave the

following fractions: hexane-ethyl acetate 3:1 (F1-F4); 2:1 (F5-F7); 1:1 (F8-F9); 1:3 (F10-F11); ethyl

acetate (F12-F14); ethyl acetate-MeOH, 95:5 (F15-F17); 9:1 (F18-F19); 1:1 (F20-F21).

Thin-layer Chromatography (TLC)

The mixture of peptides was analyzed by TLC on silica gel 5/40 µ (Czechoslovakia) in a

chloroform-methanol-water mixture (65:25:4, v/v/v), the zones were visualized using the

chlorine-tolidine reaction.

High Pressure Liquid Chromatography (HPLC)

The combined fractions F10-F17 were separated by reversed phase HPLC with a Waters

instrument (USA) on a Separon SGX column (C18, 5 µ, 4.0 i. d. x 250 mm, acetonitrile-0.01%

TFA/H2O 86:14, flow rate 1.5 ml/min, detection by UV absorption at 214 nm). The individual

depsipeptides 1, 2, and 6-8 were obtained at retention times of t1=16.2, t2=20.7, t6/7=29.7, and

t8=37.6 min.

NMR Data of Peptide 1 ([D6]-Acetone) 13С NMR: 173.90 (Cδ), 173.90 (CO), 172.15 (2-CO), 171.74 (CO), 171.60 (CO), 171.14 (CO),

170.66 (Cγ), 170.25 (CO), 57.45 (α-CH), 52.40 (α-CH), 51.75 (α-CH), 51.34 (α-CH), 50.83 (α-

CH), 49.81 (α-CH), 48.54 (α-CH), 40.68 (β-CH2), 39.0 (β-CH2), 38.43 (2 β-CH2), 35.95 (β-

CH2), 31.23 (β-CH2), 29.36 (β-CH), 28.57 (γ-CH2), 27.32 (γ-CH), 24.22 (γ-CH), 24.20 (γ-CH),

24.09 (γ-CH), 22.92 (δ1-CH3), 22.88 (δ2-CH3), 22.81 (δ1-CH3), 22.64 (δ2-CH3), 21.78 (δ1-CH3),

21.63 (δ2-CH3), 21.44 (δ1-CH3), 21.39 (δ2-CH3), 19.01 (γ1-CH3), 18.1 (γ2-CH3); alkyl chain:

169.63 (C-1), 41.06 (C-2, CH2), 71.72 (C-3, CH), 33.42 (C-4, CH2), 26.70 (C-5, CH2), 29.24-

28.82 (C-6-C-10, CH2), 30.04 (iso, C-11, CH2), 29.24 (anteiso, C-11, CH2), 38.81 (iso, C-12,

CH2), 33.69 (anteiso, C-12, CH), 27.32 (iso, C-13, CH), 29.24 (anteiso, C-13, CH2), 22.42 (iso,

C-14, CH3), 11.09 (anteiso, C-14, CH3), 22.42 (iso, 13-Met), 19.16 (anteiso, 12-Met). 1H NMR:

4.71, 4.52, 4.37, 4.25, 4.12, 4.06 (2H, αCH), 2.49 t, 2.28, 1.60-1.46 (8H), 2.88 d, 2.49, 2.64 d

(βCH); 1.88-1.60 (4H), 1.60-1.46 (2H, γCH); 1.02-0.8 (10 CH3); alkyl chain: 2.69, 2.15 (C-2,

H), 5.33 (C-3, H), 1.60-1.46 (C-4, H), 1.30-1.15 (CH2)5-12, 1.15 (C-13), 1.02-0.8 (C-14-15). NH:

8.14 (d, J=6.0), 7.86 (d, J=7.2), 7.79 (t, 2H, J=7.2), 7.72 (d, J=8.5), 7.60 (d, J=6.0), 7.34 (d,

J=8.0).

NMR Data of Peptide 2 ([D6]-DMSO) 13С NMR: 173.89 ( CO), 173.89 (Cδ),-172.06 (2 CO), 171.63 (Cγ), 171.55 (CO), 171.09 (CO),

170.61 (CO), 170.13 (CO), 56.43 (α-CH), 52.29 (α-CH), 51.78 (α-CH), 51.71 (α-CH), 51.30

(α-CH), 50.74 (α-CH), 49.77 (α-CH), 40.55 (β-CH2), 39.44 (β-CH2), 38.39 (2 β-CH2), 35.91 (β-

CH2), 35.74 (β-CH), 30.00 (β-CH2), 27.28 (γ-CH), 27.12 (γ-CH2), 24.50 (γ1-CH2), 24.19 (γ-CH),

24.16 (γ-CH), 24.07 (γ-CH), 22.89 (δ1-CH3), 22.86 (δ2-CH3), 22.81 (CH3), 22.61 (CH3), 21.81

((δ1-CH3), 21.62 (δ2-CH3), 21.44 (δ1-CH3), 21.31 (δ2-CH3), 15.41 (γ2-CH3), 10.95 (δ-CH3); alkyl

chain: 169.54 (C-1), 41.05 (C-2, CH2), 71.74 (C-3, CH), 33.26 (C-4, CH2), 26.66 (С-5, CH2),

29.20-28.78 [(C-6-C-10), CH2], 29.20 (iso, C-11, CH2), 28.52 (anteiso, C-11, CH2), 39.00 (iso,

C-12, CH2), 33.66 (anteiso, C-12, CH), 27.28 (iso, C-13, CH), 29.26 (anteiso, C-13, CH2), 22.39

(iso, C-14, CH3), 11.07 (anteiso, C-14, CH3), 22.39 (iso, 13-Met), 18.98 (anteiso, 12-Met). ). 1H

NMR: 4.43 (2H), 4.24, 4.15 (4H, αCH); 2.20, 1.93, 1.63-1.39 (8H), 2.62, 2.20, 1.75 (βCH);

1.83, 1.75 (2H), 1.63-1.39 (2H), 1.32, 1.30-1.14 (γCH); 0.89-0.74 (10 CH3); alkyl chain: 2.40,

2.13 (C-2, H), 4.97 (C-3, H), 1.63-1.39 (C-4, H), 1.30-1.14 (CH2)5-12, 1.10 (C-13), 0.89-0.74 (C-

14-15). NH: 12.11 (br s, 1H), 8.22-7.88 (m, 5H), 7.53 (br s, 1H), H/D exchangeable.

NMR Data of Peptides 6-8 have not been shown

Amino Acid Sequence Analysis The peptides 1, 2, and 6-8 were O, N-permethylated according to Hakomori’s methylation

procedure (Hakomori, 1964) after the lactone ring had been cleaved with 10% NaOH in MeOH

at room temperature for 16 h. The derivatives obtained thereof were analyzed by electron impact

mass spectrometry (EIMS, LKB 9000s) at an ionization voltage of 50 V, collector current of 60

µA, and vaporizer temperature of 200-220oC.

Preparation of Methyl Ester of Peptide 2 An ether solution of diazomethane (1 ml) was added to a solution of 2 (1 mg) in methanol (0.5

ml) and the reaction mixture was allowed to stand for 30 min at - 20oC. The solvent was

evaporated.

Reduction of Peptide 2 for the Location of the Lactone Ring

A small amount of LiBH4 (on the edge of a spatula) was added to a solution of 2 (1mg) in

methanol (0.5 ml). After 12 h, the reaction was terminated by addition of 1 M HCl and the

solution was concentrated. The residue was dissolved in water (2 ml) and extracted three times

with butanol. The organic layer was evaporated in vacuo.

Determination of the C-terminal Amino Acid of the Peptides 1, 2 and 6-8.

was A solution of peptide (1 mg) in MeOH (1 ml) and 0.5 N NaOH (1 ml) was stirred for 18 h

at room temperature, thus opening the lactone ring. The dried sample was dissolved in 5 drops

of anhydrous hydrazine and carefully filled in a reaction tube, which was then сooled to – 80oC

and sealed under vacuum. The tube was kept at 100oC for 8 h. After carefully opening the tube,

the hydrazine was evaporated in vacuo and the sample kept for 12 h in a desiccator over H2SO4.

The sample was then dissolved in 1 ml H2O together with 50 mg of glass beads (Ø 0.2 mm).

After addition of 6 drops of iso-butyraldehyde, the whole intensively shaken for 45 min. The

organic phase was 5 times extracted with ether to remove the resulting azomethines and excess

aldehyde. The remaining water phase containing the C-terminal amino acid was separated from

the glass beads and carefully dried under vacuum. For dansylation the sample was dissolved in 2

drops of 0.05 N NaHCO3-solution, 3 drops of a freshly prepared yellow solution of dansyl

chloride in acetone (2.7 mg/ml) were added and the mixture was stirred for 30 min at 37oC.

From this solution the 2D-TLC experiments for the detection of the C-terminal amino acid were

performed on polyamide (DC-Micropolyamide foils F-1700, 3 x 3 cm, Schleicher & Schull,

Germany; 1st dimension: H2O/4% formic acid, 2nd dimension: toluene/20% acetic acid). The Rf

values were compared with those of the dansylated reference amino acids valine, isoleucine,

aspartic acid, glutamic acid, and leucine.

Stereochemistry of the Amino Acids

The reference amino acids (3-5 mg) and the hydrolyzed peptides (1-2 mg) were esterified by

boiling with 5 ml iso-propanol under reflux for 2 h while the solution was saturated with gaseous

HCl. The sample was evaporated in vacuo and the residue later dissolved in 4 ml

dichloromethane. For trifluoracetylation, 1 ml of trifluoracetanhydride was added and the

mixture stirred for 3 h at room temperature. After careful evaporation to dryness, the sample was

again dissolved in 2 ml dichloromethane and subjected to GC (Siemens Sichromat 1,

split/splitless, gas/liquid injector Ø 0.3 cm, FID-column: Permabond®-L-Chiralsil-Val

(Macherey-Nagel, Dueren, Germany) 25 m x 0.32 mm, eluent gas helium, gas for FID: artificial

air/hydrogen).

RESULTS

A chloroform-methanol extract of bacterial cells was concentrated in vacuo and

chromatographed on a silica gel column using a hexane-ethyl acetate-methanol gradient as the

eluent (see experimental). FABMS analysis of the combined fractions F10-F17 revealed that it

was a mixture with quasi-molecular ions at m/z=1036, 1050, 1064, 1078 ([M+H]+), and m/z

=1058, 1072, 1086, 1100 ([M+Na]+). The 1H NMR spectrum of F10-F17 in pyridine (data not

shown) confirmed the presence of a long aliphatic chain (CH2 at δ=1.24-1.5) and a peptide

backbone by 7 NH signals at 8.7-9.6 and 7 CH protons between δ=4.5-5.2. A resonance at

δ=5.65 indicated the presence of a further proton next to oxygen, and CH3 groups gave signals at

δ=0.85-1.2.

By reversed phase HPLC (data not shown), fraction F10-F17 was found to be a mixture of

at least 8 compounds. Preparative separation by HPLC afforded substances 1 (11.4 % based on

the total fraction), 2 (42.5 %), 3 (2.1 %), 4 (2.4 %), 5 (4.2 %), 6+7 (12.6 %), and 8 (20.9 %).

Compounds 3, 4, and 5 were not analyzed because of insufficient material. The UV spectra of all

compounds showed no characteristic signals other than end absorption. The IR spectra of the

main components were identical and gave prominent broad peaks at 3300, 1646-1652, and 1538

cm-1, consistent with the presence of amide carbonyl groups, and a smaller peak at 1733 cm-1,

indicative for an ester carbonyl. Signals at wavenumbers of 2963-2858 resulting from the C-H

stretching mode suggested the presence of an aliphatic chain. Ninhydrin assay performed on 1, 2,

6-8 compounds gave a negative reaction. However, the reaction was positive after acid

hydrolysis, indicating the presence of lipopeptides with a blocked N-terminus. This result is

consistent with the above data, the behavior of studied compounds on TLC and their lipophilic

character. After total hydrolysis amino acid analysis revealed seven amino acids, 1 Asx, 1 Glx, 4

Leu, and 1 Val or Ile.

Each of the compounds 1, 2, 6-8 gave signals for 10 carbonyl groups in their 13C-NMR

spectra, 9 of which were attributed to amino acids thus confirming an acyl residue which is

contributing the 10 carbonyl. A signal at δ=71.7 in the spectra 2 (corresponding to δH=4.97,

[D6]DMSO), is characteristic for β-hydroxy fatty acids, whereas CHOH signals of α-hydroxy

acids are expected close to δ=80. The β-hydroxy group was additionally confirmed by an ABX

spin system of the acyl α-CH2 group, consisting of a pair of doublets of doublets centered at

δ=2.40 which couples with the 1H multiplet at δ=4.97. The other peptides gave similar signals.

As in all 1H-NMR spectra only 7 signals were assignable to amide NH protons thus excluding

Gln and Asn, the variations in the molecular weights of the isolated peptides represents changes

in the number of CH2 groups in the lipid and/or peptide portion (Fig. 1 and 2). Analysis of the

1H-NMR spectra of compounds studied has led us to a suggestion of their structural relatedness

with surfactins from B. subtilis (Arima et al., 1968; Hosono and Suzuki, 1983; Oka et al., 1993).

To confirm the presence of a lactone linkage, the intact peptide 2 and its alkaline

hydrolysis product were reduced with LiBH4 and the amino acid composition of the products was

analyzed. Under these conditions, ester bonds are cleaved while the peptide bond is not affected.

The reduction product of the native peptide 2 was found to lack Ile, while the ring-opened linear

peptide obtained from 2 afforded the same amino acid composition as the original compound.

When esterified with diazomethane prior to reduction with reduced LiBH4, also Asp and Glu had

disappeared thus confirming their free carboxy groups. Based on these results, it was clearly

demonstrated that the lactone ring in 2 was closed between the carboxyl group of an C-terminal

Ile residue and the β-hydroxy group of the fatty acid moiety.

The identity of the C-terminal acids in the peptides 1, 2, and 6-8 was further confirmed

by hydrazinolysis. On heating with anhydrous hydrazine, peptide bonds are cleaved forming

hydrazides from the inner acids and releasing the unchanged free C-terminal acid. The

hydrazides were removed by transformation into extractable azomethines, and the C-terminal

acid was dansylated and identified by 2D-chromatography on polyamide and comparison with

reference compounds.

Peptide 1 shows the smallest molecular weight of all isolated compounds (1035) and

carries C-terminal Val. An identical amino acid composition of Glu-Asp-Leu-Ile (1:1:4:1) was

obtained for peptides 2 and 8, and correspondingly with Ile, their 13C-spectra showed an α-

carbon signal at δ=56.43 instead at 57.45 for Val. Their molecular masses differed by ∆m=28

Da, corresponding to C15 and C17 β-hydroxy acids (M=1049 and 1077). Compounds 6 and 7

were found to be a mixture with [M+H]+ 1064, having C-terminal Ile and Val, respectively, in a

ratio of 2:1.

The peptide sequence was determined via the mass fragmentation pattern of the

permethylated peptides. The fragmentation pattern of peptides 1 and 2 were identical (Fig. 1).

The presence of peaks at m/z=412, 380 (M-MeOH) indicated that Glu is directly linked with the

C15-fatty acid moiety (Kalinovskaya et al., 1995). Signals at m/z=793, 761 (-MeOH)

demonstrated, that instead of the Val4 residue responsible for m/z=779, 747 (-MeOH) in

surfactin (molecular mass 1035; Kalinovskaya et al., 1995; Arima et al., 1968), peptides 1 and 2

must have an amino acid with one additional CH2 group, i. e. Leu4. The molecular ion peaks of

permethylated 1 and 2 (m/z =1207 and 1221) confirmed that these peptides have only different

C-terminal amino acids (Val for 1 and Ile for 2). The sequence of the ring-opened 1-acid was

found to be FA-Glu-Leu-Leu-Leu-Asp-Leu-Val. The sequence of all other components is the

same, except that the C-terminal acid in 2 and 8 is Ile, and the mixture of 6+7 was found to

contain both terminal Val and Ile.

The presence of fragment ions of peptide 8 (Fig. 1) of 28 Da higher [e. g. m/z=440, 408 (-

MeOH)] than those of peptide 2 confirmed that 8 is a homologue of 2 containing a C17- instead

of a C15-β-hydroxy acid moiety as in 2. The mass spectrum of the mixture of permethylated

peptides 6 and 7 is complex, showing two pairs of overlapping fragmentation patterns. The first

series corresponds to the permethylated acids, which are mixtures of C16- and C17-homologues

(m/z=426 and 440 etc.). The second pair originates from molecules which have lost methanol

from the (methylated) acyl constituent. The fragment ions at m/z=1077 and 1091 correspond to

the loss of the N-methylated C-terminal amino acid surmise that this is Ile in 6, whereas the

peptide 7 must have a Val7 residue, as it was previously found by dansylation.

13C NMR chemical shifts and the corresponding DEPT spectra confirmed the presence of

an iso-alkyl chain by CH3-signals at δ=22.4 and carry anteiso-due to methyl signals at δ=11.0

and 19.0 for peptides 1, 2, and 8, establishing the identity of the β hydroxy acid components as

mixture of the β hydroxy-iso-and anteiso-pentadecanoic acids for peptides 1 and 2, β- hydroxy-

iso-hexadecanoic acid in 6, β-hydroxy-iso-heptadecanoic acid in 7, β-hydroxy-iso-and anteiso-

heptadecanoic acid for peptide 8. The chirality of the β-carbinol position has not been

determined.

Asp and Glu carry two COOH groups in the molecule in 1,4-rsp. 1,5-positions. Although

it remains to be elucidated which one participates in the formation of the peptide backbone, it is

most probable that the 1-carboxyl groups form the peptide bonds as it is found in all related

peptides.

The chirality of the amino acids was determined by GCMS of trifluoroacetyl amino acid

methyl esters by comparison with D- and L-reference substances on a chiral column. Results are

shown in Table 1. Finally, the structure of isolated compounds is shown in Fig. 2.

DISCUSSION Our study revealed that the strain of B. pumilus isolated from the cuticula of the Far-Eastern

ascidian Halocynthia aurantium produced analogs of surfactins, closely related to daitocidins

(Koshino et al., 1988) or pumilacidins A-E (Naruse et al., 1990). In these compounds the fourth

amino acid of the peptide chain is replaced by Leu and the seventh by Val or Ile. Peptides 1 and

7 contained C-terminal Val, while peptides 2, 6, 8 have Ile at this position.

The surfactin analogs was found to be produced by different bacilli strains. Originally

isolated from the soil strain B. subtilis, standard surfactin or surfactin is a macrolide containing

the heptapeptide sequence Glu-Leu-Leu-Val-Asp-Leu-Leu and a lipid portion which is a mixture

of several β-hydroxy-fatty acids with chain length of 13-15 carbon atoms (Arima et al., 1968;

Hosono and Suzuki, 1983). Usually the β-OH-13-methyltetradecanoic acid (iC15) is the main

component. Later Kanatomo et al. (1995) revealed that Bacillus natto KMD 2311 contains at

least eight homologous depsipeptides with a n-, iso- or anteiso-β-hydroxy-fatty acid of carbon

number 13-16 as part of the ring system. The peptide portion of the eight homologues was found

to be identical with known surfactin. Baumgart et al. (1991) investigated surfactin from Bacillus

subtilis ATCC 21332 and assigned all proton signals to the amino acids. As the result, these

workers identified three heptapeptides and thus proved the existence of structural analogues of

surfactin. In two minor variants the C-terminal Leu is replaced by Val (10%) and Ile (20%).

Recently an isoform of surfactin, namely [Val7]surfactin (Peypoux et al., 1991) and also the

mono- and dimethyl esters of peptidelactones have been isolated (Kowall et al., 1998). A novel

[Ile7]surfactin, which showed anti-HIV activity and has a conformation different from the

known [Leu7]surfactin, has been isolated from B. subtilis natto (Itokawa et al., 1994).

Another surfactin group (Table 2) is made up of surfactant BL-86 (Horowitz and Griffin,

1991), halobacillin (Trischman et al., 1994), a new endothelin antagonist lipopeptide isolated

from of B. subtilis (Ohshima et al., 1994), isohalobacillin (Hasumi et al., 1995), lichenysin A

(Yakimov et al., 1999) and lichenysins G (Grangemard et al., 1999). The surfactant, produced by

B. licheniformis 86, is a mixture of lipopeptides with the major components having masses of

1008, 1022, 1036. The variations are due to changes in the lipid portion and/or the amino acid

composition. The one of the Glx and Asx for BL-86 is in the amide form (Gln or Asn) and the

other is present as free acid form (Asp or Glu). The peptide moiety of lichenisin A is composed

of Gln instead of Glu as the N-terminal amino acid, Ile as the C-terminal amino acid, and Val4,

Asp5, Leu2, Leu3 and Leu6. Isohalobacillin is a complex of two isomeric cyclic

acyldepsipeptides. These were identified as isomers of halobacillin. Each component of

isohalobacillin contains either a β-OH-i- or a β-OH-anteisoC15 fatty acid moiety in place of the

β-OH-nC15 fatty acid moiety found in halobacillin. However, assignment of carbon resonances

of the alkyl chain of halobacillin may be incorrected (C-15, δ=22.6 instead 14.09 ppm, Hosono

and Suzuki, 1983; Silverstein et al., 1981, and also Cδ for Ile, δ=21.6 instead of 11.2), and

halobacillin may be identical with one of the two isohalobacillin (containing a β-OH-iso-C15

fatty acid moiety). The new endothelin antagonist contains a β-OH-iC14 fatty acid moiety instead

of the mixture C15 fatty acids moiety in isohalobacillin. It can be assumed that the enzyme(s) that

synthesize the peptide portion of this family of lipopeptides in bacteria of genus Bacillus are

closely related, however, differ in specificity at the C-terminal amino acid of the chain as well as

selectivity towards Asp or Glu vs. Asn or Gln. Changing of the fermentative conditions might be

obtained new surfactin analogs with modified surface active properties. So, when B. subtilis S

499 was grown on a culture medium containing Ala as nitrogen source, a new variant of [Ala4]

surfactin was formed (Peypoux et al., 1994). The biosynthesis of [Leu4] and [Ile4] surfactins was

controlled by supplementation of L-Leu and L-Ile to the cultural medium (Grangemard et al.,

1997). The both variants have increased surface properties compared with that of surfactin.

In this study we extended the characterization of surfactin-like peptides from B. pumilus

of marine origin and have shown that the strain of B. pumilus associated with ascidian H.

aurantium produced only [Leu4]surfactins with two C-terminal variants bond to a β-hydroxy

fatty acid (C15-C17) under our fermentation conditions, while the cultivation of B. pumilus

associated with sponge Ircinia sp., under such conditions led to production of only standard

surfactins (Kalinovskaya et al., 1995). These variations, rather than being genetically

determined, depend on the specific B. pumilus strains. This structural diversity of biosurfactants

offers a potentially wider range of interfacial properties, some of which may be better suited for

specific applications. An understanding of the genetics for biosurfactants production will be

sufficient for their biotechnological application.

ACKNOWLEDGMENTS This work was partially supported by RFFR grants No. 00-04-48034, 00-15-97397 and by a

German BMBF grant 0310735

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Table 1. Evaluation of the Amino Acid Stereochemistry of the Peptides 1, 6, 7.

Aminoacid, tR Peptide 1 Peptide 7 (6)

Aspartic acid

D=27.12, L=27.26

L

tR: 27.23

L (L)

tR: 27.25

Glutamic acid

D=31.13, L=31.28

L

tR: 31.25

L (L)

tR: 31.24

Isoleucine

D=12.26a / 13.54

L=13.23a / 14.54

-

- (L)

tR: 14.70

Leucine

D=16.04, L=19.07

D/L 1:1

tR: 16.02/19.09

D/L 1:1 (D/L 1:1)

tR: 16.42/20.32

Valine

D=9.51, L=10.05

L

tR: 10.00

(too dilute)

a It is retention time for allo-Ile

29.36

R CHOMe

CH2CO Me

2Glu MeLeu MeLeu MeLeu Me

2Asp MeLeu MeX OMe

1, C15 412 539 666 793 936 1063 -

1207

− MeOH 380 507 634 761 904 1031 -

2, C15 412 539 666 793 936 1063 1190

1221

− MeOH 380 507 634 761 904 1031 -

6, C16 426 553 680 807 950 1077 -

-

− MeOH 394 521 648 775 918 1045 -

7, C17 440 567 694 821 964 1091 -

-

− MeOH 408 535 662 789 932 1059 -

8, C17 440 567 694 - 964 1091 1218

1249

− MeOH 408 535 662 761

(-CO)

932 1059 -

Fig. 1. EIMS fragmentation of permethylated acyldepsipeptides 1, 2, and 6-8

R CHO

CH2COGlu Leu Leu Leu Asp Leu

X

[M+H]

+ [M+Na]

+ [M-H]

- [M+Na-2H]

-

R X Formula

1 PB 1036 1058 1034 1056 C12-iso-and

anteiso

Val C53H93N7O13

2 PA 1050 1072 1048 1070 C12-iso-and

anteiso

Ile C54H95N7O13

6 PE 1064 1086 n.d. n.d. C13-iso Ile C55H97N7O13

7 PD 1064 1086 n.d. n.d. C14-iso Val C55H97N7O13

8 PC 1078 1100 1076 1098 C14-iso-and

anteiso

Ile C56H99N7O13

Fig. 2. Structures and Mass Data of Acyl Depsipeptides from Bacterium Bacillus pumilus

Associated with the Ascidian Halocynthia aurantium (PA – PE = Pumilacidins A – E, n.d. =

not determined).

99999999957.45

Table 2. Several types of natural surfactins

Peptide sequence Lipopeptide* Producing

strain 1 2 3 4 5 6 7

References

Surfactin

or bacircine

B. subtilis IAM 1213

B. pumilus (marine)

Glu Leu Leu Val Asp Leu Leu Arima et al. 1968

Kalinovskaya

et al. 1995 B. subtilis

S 499 Glu Leu Leu Val Asp Leu Val Peypoux et al.

1991 B. subtilis

natto Glu Leu Leu Val Asp Leu Ile Itokawa et al.

1994 Halobacillin

or Isohalobacillin

or

Lichenysin A

or Lichenysin G

Bacillus sp. CND-914, (marine)

Bacillus sp.

A 1238

B. licheniformi

s BAS 50

B. licheniformi

sIM 1307

Gln Leu Leu Val Asp Leu Ile Trischman et al. 1994

Hasumi et al. 1995

Yakimov et

al. 1999

Grangemard et al. 1999

Daitocidin

or

pumilacidin

or peptide 1, 7

Bacillus sp. Q-55

B. pumilus

937-B1

B. pumilus (marine)

Glu Leu Leu Leu Asp Leu Val Koshino et al. 1988

Naruse et al.

1990

Present paper

Daitocidin

or pumilacidin

or

peptide 2, 6, 8

Bacillus sp. Q-55

B. pumilus

937-B1

B.pumilus (marine)

Glu Leu Leu Leu Asp Leu Ile Koshino et al. 1988

Naruse et al.

1990

Present paper

*The major β-OH fatty acids that constitute the lipid moiety are iso, anteiso C13; iso, n C14; iso, anteiso C15; iso, n C16 and iso, anteiso C17.

Table 1. Evaluation of the Amino Acid Stereochemistry of the Peptides 1, 6, 7.

Table 2. Several types of natural surfactins

Fig. 1. EIMS fragmentation of permethylated acyldepsipeptides 1, 2, and 6-8

Fig. 2. Structures and Mass Data of Acyl Depsipeptides from Bacterium Bacillus pumilus

Associated with the Ascidian Halocynthia aurantium (PA – PE = Pumilacidins A – E, n.d. =

not determined).