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Characterization of two anti-fungal lipopeptides produced by Bacillus amyloliquefaciens SH-B10
Bioresource Technology 101 (2010) 8822–8827
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Characterization of two anti-fungal lipopeptides produced by Bacillus amyloliquefaciens SH-B10 Lili Chen a,b,1, Nan Wang a,1, Xuemei Wang a, Jiangchun Hu a,*, Shujin Wang a a b
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 21 May 2010 Accepted 4 June 2010
Keywords: Bacillus amyloliquefaciens Lipopeptide Fengycin Marine microorganisms
a b s t r a c t Bacillus amyloliquefaciens SH-B10 isolated from deep-sea sediment produces two anti-fungal lipopeptides that were purified by bioactivity-guided fractionation based on acid precipitation, vacuum flash chromatography and semi-preparative HPLC. The two compounds were identified by tandem Q-TOF mass spectroscopy as C16 fengycin A (1) and a new fengycin with an aminobutyric acid at position 6 of the peptide backbone (2). Both compounds showed significant inhibitory activities against five plant fungal pathogens in paper-agar disk diffusion assay. This is the first report on the anti-fungal activities of the rare 6-Abu fengycin lipopeptides, and at the same time provided an insight into the potential of marine microbial resource in biological control and sustainable agriculture. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Food-safety and environmental concerns associated with the use of chemical pesticides have increased interest in biological control as alternatives or a supplemental ways to suppress soilborne pathogens and reduce the use of agro-chemicals (Bale et al., 2008; Haas and Défago, 2005). There have been marked advances in biological control of soil-borne fungal phytopathogens with beneficial microorganisms (Raaijmakers et al., 2009). Most of the studies have focused on the microorganisms of terrestrial origin (Lugtenberg and Kamilova, 2009), but it has become obvious that microorganisms from marine environments can also produce metabolites that are potential biocontrol agents. Laboratory and field tests have demonstrated that marine bacteria can suppress plant fungal pathogens and restore soil microbial community structure (Li et al., 2008; Xu et al., 2009). One way by which biocontrol microorganisms lessen or prevent the deleterious effects of phytopathogenic organisms is by producing antibiotic metabolites (Lugtenberg and Kamilova, 2009). Characterization of the active metabolites has been proven to be an efficient way to find lead compounds for new pesticides (Dayan et al., 2009). In this study, the anti-fungal compounds produced by a marine bacterium identified as Bacillus amyloliquefaciens SH-B10 were isolated and identified as C16 fengycin A and a fengycin derivative
* Corresponding author. Tel.: +86 24 83970386; fax: +86 24 83970300. E-mail address: [email protected] (J. Hu). 1 Equal contribution as first authors. 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.054
with an aminobutyric acid residue within its cyclic peptide structure.
2. Methods 2.1. Microorganisms and culture conditions B. amyloliquefaciens SH-B10 (Genbank accession number: HM150666) isolated from a 3601-m deep-sea sediment collected from the South China Sea at 17°530 5900 N, 114°340 58.600 E was cultured in medium containing (g/l): sucrose 20, NH4NO3 2, KH2PO4 3, Na2HPO4 10, MgSO47H2O 0.2, yeast exact 0.2, CaCl2 7 106, MnSO44H2O 1 106, pH 7.0–7.2 (Liu et al., 2008). Seed cultures were prepared with two 500-ml Erlenmeyer flasks each containing 100 ml of medium and cultivated at 28 °C for 48 h with shaking at 180 rpm. A 10 l fermenter (East Biotech, China) was used for largescale production of lipopeptides in the same medium under conditions of an inoculation volume of 3% (v/v), a temperature of 30 °C, an aeration rate of 1.7 v/v/min, an agitation speed of 180 rpm and no initial pH control. Five soil-borne phytopathogens, namely Fusarium oxysporum f. sp. cucumerinum, Fusarium graminearum, F. oxysporum f. sp. vasinfectum, F. oxysporum f. sp. cucumis melo L. and F. graminearum f. sp. zea mays L. were maintained on potato sucrose agar (PSA). F. oxysporum f. sp. cucumerinum was used as target pathogen for bioactivity-guided fractionation and purification. Specimens of the strains mentioned above were deposited at the Group of Microbial Biotechnology, Institute of Applied Ecology, Chinese Academy of Sciences.
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2.2. Paper-agar disk diffusion assay Pure compounds or fractions were dissolved in phosphate buffered saline (PBS) (pH 8.5) to a concentration of 2 mg/ml and twofold serially dilution to final concentrations of 1000, 500, 250, 125 lg/ml. For the paper-agar disk diffusion assay, the method described by Raahave (1974) was used with minor modifications. Ten microliters of the solutions were applied to 8-mm filter paper disks which were evenly placed on the PSA agar plates individually inoculated with the five plant pathogens. Ten microliters of PBS (pH 8.5) was used as blank. Each concentration was assayed in triplicate and the diameters of inhibition zone were measured with a slide gauge. 2.3. Preparation of crude lipopeptides After fermentation at 28 °C for 48 h, the culture broth of SH-B10 was adjusted to pH 8.0 and centrifuged at 4000g for 30 min. The supernatant was adjusted to pH 2.0 with 6 mol/l HCl and stored overnight at 4 °C. The precipitate was recovered by centrifugation at 4000g for 20 min, washed twice with deionized water (adjusted to pH 2.0 with 6 mol/l HCl), and extracted three times with methanol. The extracts were combined and evaporated at 30 °C under vacuum to dryness with a rotary evaporator to yield pale-yellow crude lipopeptides. 2.4. Vacuum flash chromatography (VFC) The crude lipopeptides (8.28 g) obtained from 10 l of fermentation broth were subjected to vacuum flash chromatography over silica gel (600–800 mesh) and eluted with eight mixtures of dichloromethane and methanol at ratios of (v/v) 100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 50:50 and 0:100. 2.5. Reversed-phase high performance liquid chromatography (RPHPLC) purification Anti-fungal activity was found in the fraction (yield: 673.6 mg, 8.1%) obtained by elution with dichloromethane/methanol (80:20, v/v). A portion (368.0 mg) of this fraction was further separated by a semi-preparative HPLC system (Dionex U3000, USA) using a C18 YMC-Pack ODS-A column (5 lm, ø10 250 mm) eluting with 60% acetonitrile containing 0.05% trifluoroacetic acid (TFA) at a flow rate of 2.5 ml/min with UV detection at 210 nm. 2.6. Tandem (Q-TOF) mass spectrometry ESI-Q-TOF MS/MS experiments were performed with a Waters Micromass Q-TOF 2 spectrometer equipped with a nanoflow-ESI interface, a quadrupole MS-1 mass analyzer, a hexapole collision cell, and an orthogonal acceleration Time-of-Flight MS-2 mass analyzer. Argon was used as collision gas. Operating parameters included capillary voltage of 800 V, cone voltage of 50 V and orthogonal acceleration voltage of 9.1 kV. 2.7. Identification of the fatty acid (FA) side chain of 2 with GC–MS Two mg of compound 2 yielded from semi-preparative HPLC (retention time: 11.98 min) was hydrolyzed with 6 mol/l hydrochloric acid at 110 °C for 24 h in a sealed 4-ml vial. The free fatty acid was obtained after three extractions with 4 ml of chloroform and drying under a nitrogen gas stream. The residue was dissolved in 4 ml of methanol containing 1% sulfuric acid (v/v) and incubated at 70 °C for 1 h. The reaction mixture was cooled to room temperature and extracted three times with 4 ml of hexane. The hexane
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extract was dried under a stream of nitrogen gas, and the fatty acid methyl ester was subjected to GC–MS analysis. A Trace GC Ultra™-Trace MS (Thermo Scientific, USA) GC–MS system (EI source) coupled with an AS 2000 autosampler and an AS 2000 controller was used for GC–MS analysis on a DB-5 ms GC column (30 m 0.24 mm, 0.25 lm; Agilent, USA) with He as carrier gas without flow split (0.6 ml/min). The injector and source temperature were 250 and 200 °C, respectively. The oven temperature was maintained at 140 °C for 3 min, then raised at a rate of 4 °C/min to a final temperature of 230 °C. The detector voltage was 350 V. 3. Results and discussion 3.1. Bioactivity-guided isolation and purification Fermentation broth of SH-B10, crude lipopeptides, fractions from VFC and pure compounds obtained from HPLC separation were assayed with paper-agar disk diffusion test, as described above. It was found that the anti-fungal constituents could readily be precipitated from the acidified fermentation broth. After fractionation of the crude lipopeptides with VFC, the anti-fungal activity against F. oxysporum f. sp. cucumerinum was found in the fraction obtained by elution with dichloromethane/methanol (80:20 v/v) which finally afforded two active pure compounds eluted at 11.23 min (1, 55.5 mg) and 11.98 min (2, 38.2 mg) from semi-preparative HPLC purification. 3.2. Structure elucidation of compound 1 In low resolution ESI MS experiments, 1 and 2 produced ion peaks with mass difference of 14 Da at m/z 1464.1 and 1478.1, respectively, indicating that the two compounds might be structural analogs with different side chain length (–CH2–). The full scan spectrum (MS1) for compound 1 gave singly- and doubly-protonated molecular ions at m/z 1463.7069 [M+H]+ and m/z 732.3621 [M+2H]2+, respectively. The latter was chosen as precursor ion for further tandem MS fragmentation. The resulting spectrum mainly consisted of b- and y-type ions (Fig. 1A). Two predominant fragment ions at m/z 1080.56 (base peak, ion with neutral loss of b-hydroxy fatty acid-Glu) and 966.48 (ion with neutral loss of bhydroxy FA-Glu-Orn) were characteristic for fengycin A (Ala at position 6) (Fig. 2). Low mass fragments found as immonium ions at m/z 136.08 and m/z 101.07 indicated the presence of Tyr and Gln in the peptide sequence, which could also serve as a proof for confirming that a Gln rather than a Lys was substituted at position 8 in the peptide backbone, because Lys was supposed to yield an immonium ion at m/z 101.11. A cleavage at the C-terminus of Ile10 produced a linear acylium ion for further fragmentation (Fig. 3A). Successive fragmentations from the two termini of this linear peptide acylium resulted in btype ions at m/z 1350.73 (b10), 1187.70 (b9), 1059.56 (b8), 962.58 (b7), 891.54 (b6), 762.47 (b5), 661.44 (b4), 498.37 (b3) and 384.29 (b2), along with corresponding y-type ions detected at m/z 1209.59 (y10), 1080.56 (y9), 966.48 (y8), 803.42 (y7), 573.35 (y5), 502.28 (y4), and 277.16 (y2). These fragment ions allowed assignment of the peptide sequence, Glu1-Orn2-Tyr3-Thr4Glu5-Ala6-Pro7-Gln8-Tyr9-Ile10 in the structure (Fig. 3A). Ring-opening at the N-terminus of Thr4 produced another linear acylium ion (Fig. 3B). A series of dominant y-type fragments at m/z 1362.80 (y0 7), 1233.73 (y0 6), 1162.69 (y0 5), 1065.68 (y0 4), 937.59 (y0 3), 774.52 (y0 2), 661.44 (y0 1) and corresponding b-type fragment ions at m/z 102.06 (b0 1), 231.10 (b0 2), 302.14 (b0 3), 527.26 (b0 5), 690.32 (b0 6), 803.42 (b0 7) were ascribable to a peptide sequence of Tyr3-Ile10-Tyr9-Gln8-Pro7-Ala6-Glu5-Thr4 (Fig. 3B).
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Fig. 1. Positive ESI-Q-TOF MS2 spectra resulting from precursor ion of m/z 1463.81 [M+1]+ (A) and precursor ion of m/z 739.42 [M+2H]2+ (B).
Proline is always associated with very abundant fragment ions (Ioannis, 1995). As found by Vater et al. (2002), decyclization of fengycin A at the N-terminus of Pro7 could lead to a T-like structure, as revealed by a specific series of proline-directed ions. It appeared that further cleavages of the branched acylium ion might have occurred in two different ways and thus resulted in different
ions as illustrated in Fig. 3C and D. Successive loss of amino acid residues by cleavages at the peptide side chain A–E–T followed by successive cleavages at the other peptide side chain P–Q–Y–I led to the production of ions shown in Fig. 3C. In the other fragmentation pattern, cleavages only occurred from the terminus of the side chain P–Q–Y–I (Fig. 3D). In addition, peptides with proline
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Fig. 2. Structures of compound 1 and 2 characterized from the fermentation broth of B. amyloliquefaciens SH-B10.
at neither the N- nor the C-terminus often yield abundant internal fragment ions (Ioannis, 1995). A y-type cleavage at the N-terminus of Orn2 and consequential y-type loss of amino acid residues from the side chain P–O–Y–I in the branched acylium ion gave a series of unique internal fragment ions at m/z 983.52, 855.45, 692.39 and 579.29 (Fig. 3E). Taken together, compound 1 was unequivocally identified as C16 fengycin A (Fig. 2). 3.3. Structure elucidation of compound 2 In the full scan spectrum (MS1) of compound 2, a doubly-protonated molecular ion was found at m/z 739.42 [M+2H]2+ and was used as precursor ion for MS2 analysis. In the tandem MS spectrum of compound 2, similar with those of compound 1, ion peaks with high intensities at m/z 1477.85, 1459.85, 1449.87 could be assigned to singly-pronated molecular ions of [M+H]+, [M+HH2O]+ and [M+HCO]+, respectively, whereas doubly-protonated ions which were absent in the MS2 spectrum of compound 1 were found to be of high abundances at m/z 739.32 [M+2H]2+ and m/z 721.29 [M+2HCO]2+ (Fig. 1B). The base peak found at m/z 1094.58 and another y-type ion at m/z 980.51 which were 14 Da more than those of compound 1, showed that compared with 1, 2 might have an additional methene group in the peptide ring moiety instead of in the FA side chain. This was confirmed by the identical fragmentation ions derived from FA side chain at m/z 254.26 (FA), 384.29 (FA-Glu) and m/z 498.38 (FA-Glu-Orn). The pair of fragmentation ions resulting from cleavages at either of the ter-
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mini of Orn2 residue in fengycins are diagnostically important, as m/z 1080 and 966 are clear indications for fengycin A and m/z 1108, 994 for fengycin B. Apparently, in the case of compound 2, m/z 1094 and m/z 980 may stand for a new class of fengycin derivatives. Peptide ring-opening of compound 2 was favored either at the N-terminus of Pro7 or at the N-terminus of Thr4. The former peptide bond scission and sequential amino acid residues cleavage off one by one from the peptide side chain P–Q–Y–I gave rise to y-type fragment ions at m/z 1252.76, 1089.70 and m/z 661.45 as well as btype ions at m/z 226.12 and m/z 389.19 (Fig. 4B). Similarly, distinct internal fragmentations were recognized at m/z 1094.58, 869.48, 706.41 as y-type ions (Fig. 4C). These data clearly demonstrated that there was no chemical modification in the peptide side chain P–Q–Y–I including the joint Tyr3 residue and the FA side chain. Accordingly, chemical modification must have occurred in one of the three amino acid residues in peptide side chain X–E–T. The latter peptide bond scission mentioned above produced a linear peptide acylium ion which was further cleaved by loss of successive amino acid residues from both termini to give two series of ions as shown in Fig. 4A. The resulting b-type fragment ions at m/z 1376.83, 1247.64, 1162.73 as well as y-type fragment ions at m/z 231.10 and m/z 413.28 revealed an unambiguously determined side chain sequence of Abu-Glu-Thr. Different FA termini (normal-, iso- and anteiso-) produce carbenium ions with different stability in EI MS experiments, and consequently display different relative abundance levels of the fragment ions derived from FA terminus in EI MS spectra. The relative abundance ratio of fragment m/z 43 to m/z 57 (I43/I57) can serve as an indicator for the FA terminal structure type (Yang et al., 2007). In GC–MS experiments, the methyl ester of the FA moiety of 2 was detected at 19.05 min in GC chromatogram and an I43/I57 value of 1.3 was found in the EI MS spectrum, showing that 2 possesses a normal-type FA side chain. Furthermore, searching of the authentic EI MS spectra library installed in the GC–MS system also led to the identification of the compound as methyl 3-hydroxyhexadecanoate. Therefore, compound 2 was defined as a new n-C16 fengycin with an Abu substitution at position 6 (Fig. 2).
Fig. 3. Fragmentation patterns produced from singly protonated precursor ion of m/z 1463.81 [M+1]+ for compound 1. Peptide ring-opening occurred at the C-terminus of Ile10 (A). Ring-opening occurred at the N-terminus of Thr4 (B). Peptide ring-opening occurred at the N-terminus of Pro7 to yield a branched acylium ion, followed by subsequent cleavages in peptide side chain A–E–T, and then in side chain P–Q–Y–I (C). Cleavages occurred only in the side chain P–Q–Y–I (D). And y-type cleavage at the Nterminus of Orn and ring-opening cleavage at the N-terminus of Pro7 resulted in another branched acylium ion which was subjected to further internal fragmentations in side chain P–Q–Y–I (E).
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Fig. 4. Fragmentation patterns produced from doubly-protonated precursor ion of m/z 739.42 [M+2H]2+ for compound 2. Peptide ring-opening occurred at the N-terminus of Thr4 (A). Peptide ring-opening occurred at the N-terminus of Pro7 to yield a branched acylium ion, followed by subsequent cleavages in peptide side chain P–Q–Y–I (B). And y-type cleavage at the N-terminus of Orn and ring-opening cleavage at the N-terminus of Pro7 resulted in another branched acylium ion which was subjected to further internal fragmentations in side chain P–Q–Y–I (C).
Table 1 In vitro anti-fungal activities of compound 1 and 2 against five soil-borne fungal phytopathogens by paper-agar disk diffusion tests. Compound
1 2 1 2 1 2 1 2 1 2 Control a b
Conc. (mg/ ml)
Diameter of inhibition zone (mm)a,b F. oxysporum f. sp. cucumerinum
F. graminearum
F. oxysporum f. sp. cucumis melo L.
F. oxysporum f. sp. vasinfectum
F. graminearum f. sp. zea mays L.
2
20.49 ± 0.96 16.01 ± 0.16 18.48 ± 1.83 13.11 ± 0.78 15.93 ± 1.51 10.76 ± 1.94 14.80 ± 0 9.45 ± 0.96 8 8 8
18.52 ± 1.88 14.51 ± 0.50 15.93 ± 0.06 13.69 ± 0.32 12.47 ± 0.99 10.84 ± 1.00 10.84 ± 0.68 9.37 ± 0.30 8 8 8
18.15 ± 0.43 14.24 ± 0.82 15.25 ± 0.57 11.41 ± 0.49 8 8.64 ± 1.11 8 8 8 8 8
14.2 ± 0.55 16..23 ± 0.58 14.55 ± 0.95 14.53 ± 0.74 9.99 ± 0.86 10.60 ± 0.40 9.36 ± 0.28 9.24 ± 0.31 8 8 8
22.71 ± 1.75 15.43 ± 0.49 19.66 ± 0.82 12.47 ± 1.25 17.59 ± 1.49 10.21 ± 0.67 11.88 9.09 ± 0.36 8 8.68 ± 0.08 8
1 0.5 0.25 0.125
Diameter of inhibition zone represent the mean ± SD of three independent readings; SD: standard deviation. Diameter of paper disks: 8 mm.
3.4. The anti-fungal activities of 1 and 2 Both 1 and 2 showed significant inhibitory activities against the tested plant pathogens in a concentration-dependent manner (Table 1). Generally, 1 demonstrated a little better inhibitory activities than 2, but the difference was not statistically significant. The minimum inhibitory concentrations of 2 against F. oxysporum f. sp. cucumis melo L. and F. graminearum f. sp. zea mays L. were apparently lower than 1. 3.5. Discussion Fengycins are versatile lipodecapeptides applicable in biocontrol of plant pathogens (Chan et al., 2009) as well as mosquito larvae (Das and Mukherjee, 2006), as anti-adhesion agents against biofilm (Rivardo et al., 2009), and as biosurfactants for polycyclic aromatic hydrocarbons (PAHs) degradation (Das et al., 2008). When used to suppress phytopathogenic fungi, fengycins are capable of altering the structure and permeability of cell membrane (Deleu et al., 2005). In addition to direct antipathogenic action, they also serve as determinants in triggering induced systemic resistance (ISR) in plant systems (Romero et al., 2007). Compared with iturins and surfactins, fengycins are equally widespread in
occurrence but relatively low in structure diversity regarding their peptide moiety. Fengycin A and fengycin B have been described as the only two main types of structural variants which differ only in the amino acid residue at position 6, namely fengycin A has an Ala at position 6, in contrast to fengycin B which has a Val at the same position. A large number of previous studies on the structure of fengycins dealt with the fengycins homologs varying in fatty acid side chain length. But Kimura (1997), Esumi (2003) and Pueyo (2009) found several new fengycins variants produced by Bacillus bacteria in which Ile was changed to Val at the position 10 of the peptide moiety. Previously, the uncommon 6-Abu fengycins isolated from a soil bacterium were recognized as plipastatin species (Kimura et al., 1997; Esumi et al., 2003). In tandem ms spectrum, 2 showed characterized ion peaks at m/z 1094.6 and 980.5, which is clearly different with fengycin A and B. Hu et al. (2007) reported that a precursor ion of m/z 1479.0 could afford product ions at m/z 1094.6 and 980.5 in HPLC–ESI/CID (collision induced dissociation) Mass experiments and claimed that it was a fengycin derivative. For the first time, we fully characterize a 6-Abu substituted fengycin variant based on intensive Q-TOF mass spectroscopic structure elucidation. HPLC-tandem mass has been proved to be very applicable for both quantification and qualification of fengycins (Kinsella et al.,
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2009; Vater et al., 2002). Product ions arising from amide-bond scission at either of the termini of Orn2, the most basic amino acid residue in the structure, are the most decisive and informative for the determination of the structure types of fengycins. These ornithine-derived ions can serve as good indicators for basic structure type judgements. This also implies that it is not always safe to deduce fengycin analogs from the quasi-molecular ions differences, because fengycin isomers may produce distinct product ions even though their quasi-molecular ions are exactly same. Ring-opening sites in mass experiments are also very important for sequencing of cyclic lipopeptide backbone. Basically, as a better hydrogen bond receptor, the N-terminus of proline residue at position 7 is frequently involved in initial cleavage of peptide ring, resulting in a branched acylium ion (Vater et al., 2002). Ring cleavages have also been reported to occur at the C-terminus of Ile10, the only ester bond in the structure, to produce a linear acylium ion which will be subjected to further fragmentation (Hagelin et al., 2007). In this paper, Fig. 3B and A depicts an additional ring cleavage pattern: amide-bond cleavage in Tyr3 residue leading to another linear acylium ion. In addition, internal cleavages associated with fragmentations of the peptide backbones of fengycins could also provide valuable sequencing information for structure elucidation. Despite still being an emerging field, marine microorganisms and their biologically active metabolites have become hot resources for new drug candidates and lead compounds (Fenical, 1993). Laboratory and field tests have shown that marine-organism derived biocontrol agents can potentially control soil-borne fungal phytopathogens attacking cucumber, soy bean, chestnut, cotton and Chinese fir plantation (Li et al., 2008; Xu et al., 2009). Given the significant phytopathogen inhibitory activities of fengycins, 6-Abu fengycin together with other Bacillus lipopeptides can potentially find applications in biocontrol and sustainable agriculture.
4. Conclusions B. amyloliquefaciens SH-B10 produces the lipopeptides C16 fengycin A and a new derivative containing aminobuturic acid instead of alanine at position 6 of the peptide. The derivative also has anti-fungal properties. These findings illustrate that marine-derived organisms and their metabolites have potential as biorational pesticides and biocontrol agents.
Acknowledgements This work was supported by Grants from the National High Technology Research and Development Program of China (863 Program) (No. 2007AA09Z417), Chinese Academy of Sciences Innovation Projects (No. KZCX2-YW-209) and Network Laboratory for Coastal Zone Microbial Research, CAS (No. KSCX2-YW-G-073). We also want to acknowledge Ms. Ping Li of the National Center of Biomedical Analysis at Academy of Military Medical Sciences for assistance in Mass experiments.
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Characterization of two anti-fungal lipopeptides produced by Bacillus amyloliquefaciens SH-B10 Lili Chen a,b,1, Nan Wang a,1, Xuemei Wang a, Jiangchun Hu a,*, Shujin Wang a a b
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 21 May 2010 Accepted 4 June 2010
Keywords: Bacillus amyloliquefaciens Lipopeptide Fengycin Marine microorganisms
a b s t r a c t Bacillus amyloliquefaciens SH-B10 isolated from deep-sea sediment produces two anti-fungal lipopeptides that were purified by bioactivity-guided fractionation based on acid precipitation, vacuum flash chromatography and semi-preparative HPLC. The two compounds were identified by tandem Q-TOF mass spectroscopy as C16 fengycin A (1) and a new fengycin with an aminobutyric acid at position 6 of the peptide backbone (2). Both compounds showed significant inhibitory activities against five plant fungal pathogens in paper-agar disk diffusion assay. This is the first report on the anti-fungal activities of the rare 6-Abu fengycin lipopeptides, and at the same time provided an insight into the potential of marine microbial resource in biological control and sustainable agriculture. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Food-safety and environmental concerns associated with the use of chemical pesticides have increased interest in biological control as alternatives or a supplemental ways to suppress soilborne pathogens and reduce the use of agro-chemicals (Bale et al., 2008; Haas and Défago, 2005). There have been marked advances in biological control of soil-borne fungal phytopathogens with beneficial microorganisms (Raaijmakers et al., 2009). Most of the studies have focused on the microorganisms of terrestrial origin (Lugtenberg and Kamilova, 2009), but it has become obvious that microorganisms from marine environments can also produce metabolites that are potential biocontrol agents. Laboratory and field tests have demonstrated that marine bacteria can suppress plant fungal pathogens and restore soil microbial community structure (Li et al., 2008; Xu et al., 2009). One way by which biocontrol microorganisms lessen or prevent the deleterious effects of phytopathogenic organisms is by producing antibiotic metabolites (Lugtenberg and Kamilova, 2009). Characterization of the active metabolites has been proven to be an efficient way to find lead compounds for new pesticides (Dayan et al., 2009). In this study, the anti-fungal compounds produced by a marine bacterium identified as Bacillus amyloliquefaciens SH-B10 were isolated and identified as C16 fengycin A and a fengycin derivative
* Corresponding author. Tel.: +86 24 83970386; fax: +86 24 83970300. E-mail address: [email protected] (J. Hu). 1 Equal contribution as first authors. 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.054
with an aminobutyric acid residue within its cyclic peptide structure.
2. Methods 2.1. Microorganisms and culture conditions B. amyloliquefaciens SH-B10 (Genbank accession number: HM150666) isolated from a 3601-m deep-sea sediment collected from the South China Sea at 17°530 5900 N, 114°340 58.600 E was cultured in medium containing (g/l): sucrose 20, NH4NO3 2, KH2PO4 3, Na2HPO4 10, MgSO47H2O 0.2, yeast exact 0.2, CaCl2 7 106, MnSO44H2O 1 106, pH 7.0–7.2 (Liu et al., 2008). Seed cultures were prepared with two 500-ml Erlenmeyer flasks each containing 100 ml of medium and cultivated at 28 °C for 48 h with shaking at 180 rpm. A 10 l fermenter (East Biotech, China) was used for largescale production of lipopeptides in the same medium under conditions of an inoculation volume of 3% (v/v), a temperature of 30 °C, an aeration rate of 1.7 v/v/min, an agitation speed of 180 rpm and no initial pH control. Five soil-borne phytopathogens, namely Fusarium oxysporum f. sp. cucumerinum, Fusarium graminearum, F. oxysporum f. sp. vasinfectum, F. oxysporum f. sp. cucumis melo L. and F. graminearum f. sp. zea mays L. were maintained on potato sucrose agar (PSA). F. oxysporum f. sp. cucumerinum was used as target pathogen for bioactivity-guided fractionation and purification. Specimens of the strains mentioned above were deposited at the Group of Microbial Biotechnology, Institute of Applied Ecology, Chinese Academy of Sciences.
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2.2. Paper-agar disk diffusion assay Pure compounds or fractions were dissolved in phosphate buffered saline (PBS) (pH 8.5) to a concentration of 2 mg/ml and twofold serially dilution to final concentrations of 1000, 500, 250, 125 lg/ml. For the paper-agar disk diffusion assay, the method described by Raahave (1974) was used with minor modifications. Ten microliters of the solutions were applied to 8-mm filter paper disks which were evenly placed on the PSA agar plates individually inoculated with the five plant pathogens. Ten microliters of PBS (pH 8.5) was used as blank. Each concentration was assayed in triplicate and the diameters of inhibition zone were measured with a slide gauge. 2.3. Preparation of crude lipopeptides After fermentation at 28 °C for 48 h, the culture broth of SH-B10 was adjusted to pH 8.0 and centrifuged at 4000g for 30 min. The supernatant was adjusted to pH 2.0 with 6 mol/l HCl and stored overnight at 4 °C. The precipitate was recovered by centrifugation at 4000g for 20 min, washed twice with deionized water (adjusted to pH 2.0 with 6 mol/l HCl), and extracted three times with methanol. The extracts were combined and evaporated at 30 °C under vacuum to dryness with a rotary evaporator to yield pale-yellow crude lipopeptides. 2.4. Vacuum flash chromatography (VFC) The crude lipopeptides (8.28 g) obtained from 10 l of fermentation broth were subjected to vacuum flash chromatography over silica gel (600–800 mesh) and eluted with eight mixtures of dichloromethane and methanol at ratios of (v/v) 100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 50:50 and 0:100. 2.5. Reversed-phase high performance liquid chromatography (RPHPLC) purification Anti-fungal activity was found in the fraction (yield: 673.6 mg, 8.1%) obtained by elution with dichloromethane/methanol (80:20, v/v). A portion (368.0 mg) of this fraction was further separated by a semi-preparative HPLC system (Dionex U3000, USA) using a C18 YMC-Pack ODS-A column (5 lm, ø10 250 mm) eluting with 60% acetonitrile containing 0.05% trifluoroacetic acid (TFA) at a flow rate of 2.5 ml/min with UV detection at 210 nm. 2.6. Tandem (Q-TOF) mass spectrometry ESI-Q-TOF MS/MS experiments were performed with a Waters Micromass Q-TOF 2 spectrometer equipped with a nanoflow-ESI interface, a quadrupole MS-1 mass analyzer, a hexapole collision cell, and an orthogonal acceleration Time-of-Flight MS-2 mass analyzer. Argon was used as collision gas. Operating parameters included capillary voltage of 800 V, cone voltage of 50 V and orthogonal acceleration voltage of 9.1 kV. 2.7. Identification of the fatty acid (FA) side chain of 2 with GC–MS Two mg of compound 2 yielded from semi-preparative HPLC (retention time: 11.98 min) was hydrolyzed with 6 mol/l hydrochloric acid at 110 °C for 24 h in a sealed 4-ml vial. The free fatty acid was obtained after three extractions with 4 ml of chloroform and drying under a nitrogen gas stream. The residue was dissolved in 4 ml of methanol containing 1% sulfuric acid (v/v) and incubated at 70 °C for 1 h. The reaction mixture was cooled to room temperature and extracted three times with 4 ml of hexane. The hexane
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extract was dried under a stream of nitrogen gas, and the fatty acid methyl ester was subjected to GC–MS analysis. A Trace GC Ultra™-Trace MS (Thermo Scientific, USA) GC–MS system (EI source) coupled with an AS 2000 autosampler and an AS 2000 controller was used for GC–MS analysis on a DB-5 ms GC column (30 m 0.24 mm, 0.25 lm; Agilent, USA) with He as carrier gas without flow split (0.6 ml/min). The injector and source temperature were 250 and 200 °C, respectively. The oven temperature was maintained at 140 °C for 3 min, then raised at a rate of 4 °C/min to a final temperature of 230 °C. The detector voltage was 350 V. 3. Results and discussion 3.1. Bioactivity-guided isolation and purification Fermentation broth of SH-B10, crude lipopeptides, fractions from VFC and pure compounds obtained from HPLC separation were assayed with paper-agar disk diffusion test, as described above. It was found that the anti-fungal constituents could readily be precipitated from the acidified fermentation broth. After fractionation of the crude lipopeptides with VFC, the anti-fungal activity against F. oxysporum f. sp. cucumerinum was found in the fraction obtained by elution with dichloromethane/methanol (80:20 v/v) which finally afforded two active pure compounds eluted at 11.23 min (1, 55.5 mg) and 11.98 min (2, 38.2 mg) from semi-preparative HPLC purification. 3.2. Structure elucidation of compound 1 In low resolution ESI MS experiments, 1 and 2 produced ion peaks with mass difference of 14 Da at m/z 1464.1 and 1478.1, respectively, indicating that the two compounds might be structural analogs with different side chain length (–CH2–). The full scan spectrum (MS1) for compound 1 gave singly- and doubly-protonated molecular ions at m/z 1463.7069 [M+H]+ and m/z 732.3621 [M+2H]2+, respectively. The latter was chosen as precursor ion for further tandem MS fragmentation. The resulting spectrum mainly consisted of b- and y-type ions (Fig. 1A). Two predominant fragment ions at m/z 1080.56 (base peak, ion with neutral loss of b-hydroxy fatty acid-Glu) and 966.48 (ion with neutral loss of bhydroxy FA-Glu-Orn) were characteristic for fengycin A (Ala at position 6) (Fig. 2). Low mass fragments found as immonium ions at m/z 136.08 and m/z 101.07 indicated the presence of Tyr and Gln in the peptide sequence, which could also serve as a proof for confirming that a Gln rather than a Lys was substituted at position 8 in the peptide backbone, because Lys was supposed to yield an immonium ion at m/z 101.11. A cleavage at the C-terminus of Ile10 produced a linear acylium ion for further fragmentation (Fig. 3A). Successive fragmentations from the two termini of this linear peptide acylium resulted in btype ions at m/z 1350.73 (b10), 1187.70 (b9), 1059.56 (b8), 962.58 (b7), 891.54 (b6), 762.47 (b5), 661.44 (b4), 498.37 (b3) and 384.29 (b2), along with corresponding y-type ions detected at m/z 1209.59 (y10), 1080.56 (y9), 966.48 (y8), 803.42 (y7), 573.35 (y5), 502.28 (y4), and 277.16 (y2). These fragment ions allowed assignment of the peptide sequence, Glu1-Orn2-Tyr3-Thr4Glu5-Ala6-Pro7-Gln8-Tyr9-Ile10 in the structure (Fig. 3A). Ring-opening at the N-terminus of Thr4 produced another linear acylium ion (Fig. 3B). A series of dominant y-type fragments at m/z 1362.80 (y0 7), 1233.73 (y0 6), 1162.69 (y0 5), 1065.68 (y0 4), 937.59 (y0 3), 774.52 (y0 2), 661.44 (y0 1) and corresponding b-type fragment ions at m/z 102.06 (b0 1), 231.10 (b0 2), 302.14 (b0 3), 527.26 (b0 5), 690.32 (b0 6), 803.42 (b0 7) were ascribable to a peptide sequence of Tyr3-Ile10-Tyr9-Gln8-Pro7-Ala6-Glu5-Thr4 (Fig. 3B).
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Fig. 1. Positive ESI-Q-TOF MS2 spectra resulting from precursor ion of m/z 1463.81 [M+1]+ (A) and precursor ion of m/z 739.42 [M+2H]2+ (B).
Proline is always associated with very abundant fragment ions (Ioannis, 1995). As found by Vater et al. (2002), decyclization of fengycin A at the N-terminus of Pro7 could lead to a T-like structure, as revealed by a specific series of proline-directed ions. It appeared that further cleavages of the branched acylium ion might have occurred in two different ways and thus resulted in different
ions as illustrated in Fig. 3C and D. Successive loss of amino acid residues by cleavages at the peptide side chain A–E–T followed by successive cleavages at the other peptide side chain P–Q–Y–I led to the production of ions shown in Fig. 3C. In the other fragmentation pattern, cleavages only occurred from the terminus of the side chain P–Q–Y–I (Fig. 3D). In addition, peptides with proline
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Fig. 2. Structures of compound 1 and 2 characterized from the fermentation broth of B. amyloliquefaciens SH-B10.
at neither the N- nor the C-terminus often yield abundant internal fragment ions (Ioannis, 1995). A y-type cleavage at the N-terminus of Orn2 and consequential y-type loss of amino acid residues from the side chain P–O–Y–I in the branched acylium ion gave a series of unique internal fragment ions at m/z 983.52, 855.45, 692.39 and 579.29 (Fig. 3E). Taken together, compound 1 was unequivocally identified as C16 fengycin A (Fig. 2). 3.3. Structure elucidation of compound 2 In the full scan spectrum (MS1) of compound 2, a doubly-protonated molecular ion was found at m/z 739.42 [M+2H]2+ and was used as precursor ion for MS2 analysis. In the tandem MS spectrum of compound 2, similar with those of compound 1, ion peaks with high intensities at m/z 1477.85, 1459.85, 1449.87 could be assigned to singly-pronated molecular ions of [M+H]+, [M+HH2O]+ and [M+HCO]+, respectively, whereas doubly-protonated ions which were absent in the MS2 spectrum of compound 1 were found to be of high abundances at m/z 739.32 [M+2H]2+ and m/z 721.29 [M+2HCO]2+ (Fig. 1B). The base peak found at m/z 1094.58 and another y-type ion at m/z 980.51 which were 14 Da more than those of compound 1, showed that compared with 1, 2 might have an additional methene group in the peptide ring moiety instead of in the FA side chain. This was confirmed by the identical fragmentation ions derived from FA side chain at m/z 254.26 (FA), 384.29 (FA-Glu) and m/z 498.38 (FA-Glu-Orn). The pair of fragmentation ions resulting from cleavages at either of the ter-
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mini of Orn2 residue in fengycins are diagnostically important, as m/z 1080 and 966 are clear indications for fengycin A and m/z 1108, 994 for fengycin B. Apparently, in the case of compound 2, m/z 1094 and m/z 980 may stand for a new class of fengycin derivatives. Peptide ring-opening of compound 2 was favored either at the N-terminus of Pro7 or at the N-terminus of Thr4. The former peptide bond scission and sequential amino acid residues cleavage off one by one from the peptide side chain P–Q–Y–I gave rise to y-type fragment ions at m/z 1252.76, 1089.70 and m/z 661.45 as well as btype ions at m/z 226.12 and m/z 389.19 (Fig. 4B). Similarly, distinct internal fragmentations were recognized at m/z 1094.58, 869.48, 706.41 as y-type ions (Fig. 4C). These data clearly demonstrated that there was no chemical modification in the peptide side chain P–Q–Y–I including the joint Tyr3 residue and the FA side chain. Accordingly, chemical modification must have occurred in one of the three amino acid residues in peptide side chain X–E–T. The latter peptide bond scission mentioned above produced a linear peptide acylium ion which was further cleaved by loss of successive amino acid residues from both termini to give two series of ions as shown in Fig. 4A. The resulting b-type fragment ions at m/z 1376.83, 1247.64, 1162.73 as well as y-type fragment ions at m/z 231.10 and m/z 413.28 revealed an unambiguously determined side chain sequence of Abu-Glu-Thr. Different FA termini (normal-, iso- and anteiso-) produce carbenium ions with different stability in EI MS experiments, and consequently display different relative abundance levels of the fragment ions derived from FA terminus in EI MS spectra. The relative abundance ratio of fragment m/z 43 to m/z 57 (I43/I57) can serve as an indicator for the FA terminal structure type (Yang et al., 2007). In GC–MS experiments, the methyl ester of the FA moiety of 2 was detected at 19.05 min in GC chromatogram and an I43/I57 value of 1.3 was found in the EI MS spectrum, showing that 2 possesses a normal-type FA side chain. Furthermore, searching of the authentic EI MS spectra library installed in the GC–MS system also led to the identification of the compound as methyl 3-hydroxyhexadecanoate. Therefore, compound 2 was defined as a new n-C16 fengycin with an Abu substitution at position 6 (Fig. 2).
Fig. 3. Fragmentation patterns produced from singly protonated precursor ion of m/z 1463.81 [M+1]+ for compound 1. Peptide ring-opening occurred at the C-terminus of Ile10 (A). Ring-opening occurred at the N-terminus of Thr4 (B). Peptide ring-opening occurred at the N-terminus of Pro7 to yield a branched acylium ion, followed by subsequent cleavages in peptide side chain A–E–T, and then in side chain P–Q–Y–I (C). Cleavages occurred only in the side chain P–Q–Y–I (D). And y-type cleavage at the Nterminus of Orn and ring-opening cleavage at the N-terminus of Pro7 resulted in another branched acylium ion which was subjected to further internal fragmentations in side chain P–Q–Y–I (E).
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Fig. 4. Fragmentation patterns produced from doubly-protonated precursor ion of m/z 739.42 [M+2H]2+ for compound 2. Peptide ring-opening occurred at the N-terminus of Thr4 (A). Peptide ring-opening occurred at the N-terminus of Pro7 to yield a branched acylium ion, followed by subsequent cleavages in peptide side chain P–Q–Y–I (B). And y-type cleavage at the N-terminus of Orn and ring-opening cleavage at the N-terminus of Pro7 resulted in another branched acylium ion which was subjected to further internal fragmentations in side chain P–Q–Y–I (C).
Table 1 In vitro anti-fungal activities of compound 1 and 2 against five soil-borne fungal phytopathogens by paper-agar disk diffusion tests. Compound
1 2 1 2 1 2 1 2 1 2 Control a b
Conc. (mg/ ml)
Diameter of inhibition zone (mm)a,b F. oxysporum f. sp. cucumerinum
F. graminearum
F. oxysporum f. sp. cucumis melo L.
F. oxysporum f. sp. vasinfectum
F. graminearum f. sp. zea mays L.
2
20.49 ± 0.96 16.01 ± 0.16 18.48 ± 1.83 13.11 ± 0.78 15.93 ± 1.51 10.76 ± 1.94 14.80 ± 0 9.45 ± 0.96 8 8 8
18.52 ± 1.88 14.51 ± 0.50 15.93 ± 0.06 13.69 ± 0.32 12.47 ± 0.99 10.84 ± 1.00 10.84 ± 0.68 9.37 ± 0.30 8 8 8
18.15 ± 0.43 14.24 ± 0.82 15.25 ± 0.57 11.41 ± 0.49 8 8.64 ± 1.11 8 8 8 8 8
14.2 ± 0.55 16..23 ± 0.58 14.55 ± 0.95 14.53 ± 0.74 9.99 ± 0.86 10.60 ± 0.40 9.36 ± 0.28 9.24 ± 0.31 8 8 8
22.71 ± 1.75 15.43 ± 0.49 19.66 ± 0.82 12.47 ± 1.25 17.59 ± 1.49 10.21 ± 0.67 11.88 9.09 ± 0.36 8 8.68 ± 0.08 8
1 0.5 0.25 0.125
Diameter of inhibition zone represent the mean ± SD of three independent readings; SD: standard deviation. Diameter of paper disks: 8 mm.
3.4. The anti-fungal activities of 1 and 2 Both 1 and 2 showed significant inhibitory activities against the tested plant pathogens in a concentration-dependent manner (Table 1). Generally, 1 demonstrated a little better inhibitory activities than 2, but the difference was not statistically significant. The minimum inhibitory concentrations of 2 against F. oxysporum f. sp. cucumis melo L. and F. graminearum f. sp. zea mays L. were apparently lower than 1. 3.5. Discussion Fengycins are versatile lipodecapeptides applicable in biocontrol of plant pathogens (Chan et al., 2009) as well as mosquito larvae (Das and Mukherjee, 2006), as anti-adhesion agents against biofilm (Rivardo et al., 2009), and as biosurfactants for polycyclic aromatic hydrocarbons (PAHs) degradation (Das et al., 2008). When used to suppress phytopathogenic fungi, fengycins are capable of altering the structure and permeability of cell membrane (Deleu et al., 2005). In addition to direct antipathogenic action, they also serve as determinants in triggering induced systemic resistance (ISR) in plant systems (Romero et al., 2007). Compared with iturins and surfactins, fengycins are equally widespread in
occurrence but relatively low in structure diversity regarding their peptide moiety. Fengycin A and fengycin B have been described as the only two main types of structural variants which differ only in the amino acid residue at position 6, namely fengycin A has an Ala at position 6, in contrast to fengycin B which has a Val at the same position. A large number of previous studies on the structure of fengycins dealt with the fengycins homologs varying in fatty acid side chain length. But Kimura (1997), Esumi (2003) and Pueyo (2009) found several new fengycins variants produced by Bacillus bacteria in which Ile was changed to Val at the position 10 of the peptide moiety. Previously, the uncommon 6-Abu fengycins isolated from a soil bacterium were recognized as plipastatin species (Kimura et al., 1997; Esumi et al., 2003). In tandem ms spectrum, 2 showed characterized ion peaks at m/z 1094.6 and 980.5, which is clearly different with fengycin A and B. Hu et al. (2007) reported that a precursor ion of m/z 1479.0 could afford product ions at m/z 1094.6 and 980.5 in HPLC–ESI/CID (collision induced dissociation) Mass experiments and claimed that it was a fengycin derivative. For the first time, we fully characterize a 6-Abu substituted fengycin variant based on intensive Q-TOF mass spectroscopic structure elucidation. HPLC-tandem mass has been proved to be very applicable for both quantification and qualification of fengycins (Kinsella et al.,
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2009; Vater et al., 2002). Product ions arising from amide-bond scission at either of the termini of Orn2, the most basic amino acid residue in the structure, are the most decisive and informative for the determination of the structure types of fengycins. These ornithine-derived ions can serve as good indicators for basic structure type judgements. This also implies that it is not always safe to deduce fengycin analogs from the quasi-molecular ions differences, because fengycin isomers may produce distinct product ions even though their quasi-molecular ions are exactly same. Ring-opening sites in mass experiments are also very important for sequencing of cyclic lipopeptide backbone. Basically, as a better hydrogen bond receptor, the N-terminus of proline residue at position 7 is frequently involved in initial cleavage of peptide ring, resulting in a branched acylium ion (Vater et al., 2002). Ring cleavages have also been reported to occur at the C-terminus of Ile10, the only ester bond in the structure, to produce a linear acylium ion which will be subjected to further fragmentation (Hagelin et al., 2007). In this paper, Fig. 3B and A depicts an additional ring cleavage pattern: amide-bond cleavage in Tyr3 residue leading to another linear acylium ion. In addition, internal cleavages associated with fragmentations of the peptide backbones of fengycins could also provide valuable sequencing information for structure elucidation. Despite still being an emerging field, marine microorganisms and their biologically active metabolites have become hot resources for new drug candidates and lead compounds (Fenical, 1993). Laboratory and field tests have shown that marine-organism derived biocontrol agents can potentially control soil-borne fungal phytopathogens attacking cucumber, soy bean, chestnut, cotton and Chinese fir plantation (Li et al., 2008; Xu et al., 2009). Given the significant phytopathogen inhibitory activities of fengycins, 6-Abu fengycin together with other Bacillus lipopeptides can potentially find applications in biocontrol and sustainable agriculture.
4. Conclusions B. amyloliquefaciens SH-B10 produces the lipopeptides C16 fengycin A and a new derivative containing aminobuturic acid instead of alanine at position 6 of the peptide. The derivative also has anti-fungal properties. These findings illustrate that marine-derived organisms and their metabolites have potential as biorational pesticides and biocontrol agents.
Acknowledgements This work was supported by Grants from the National High Technology Research and Development Program of China (863 Program) (No. 2007AA09Z417), Chinese Academy of Sciences Innovation Projects (No. KZCX2-YW-209) and Network Laboratory for Coastal Zone Microbial Research, CAS (No. KSCX2-YW-G-073). We also want to acknowledge Ms. Ping Li of the National Center of Biomedical Analysis at Academy of Military Medical Sciences for assistance in Mass experiments.
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