Identification and characterization of novel surfactins produced by fungal antagonist Bacillus amyloliquefaciens 6B
Khyati V. Pathak Anjali Bose Haresh Keharia∗
BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, Sardar Patel University, Vallabh Vidyangar, Gujarat, India
Abstract The broad-spectrum fungal antagonist, Bacillus amyloliquefaciens 6B (BA6B), isolated from the Jakhao coast of Kutch, India, was investigated for its antifungal metabolites using mass spectrometry. The cyclic lipopeptides harvested from the cell-free fermentation broth of BA6B by acid precipitation and subsequently dissolved in methanol were subjected to liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI–MS/MS) for their identification and sequence determination. The 26 types of surfactin variants were identified from the methanolic extract
by LC–ESI–MS/MS analysis. Among 26 surfactin species, several new cyclic as well as acyclic surfactin variants based on the variation in the β-hydroxy fatty acid (β-OH FA) chain length and/or in amino acid positions 4, 5, 6, and 7 were identified. The mass spectrometric analysis of crude extract also enabled the identification of 11 unique molecular mass ions with minimum two or maximum four types of isobaric peptide variants. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 0, Number 0, Pages 1–8, 2014
Keywords: Bacillus amyloliquefaciens, surfactin, isobaric cyclic lipopeptides, mass spectrometry
1. Introduction Microbial cells are one of the most elegant chemical factories manufacturing a plethora of bioactive natural compounds to perform various physiological functions, such as growth and its regulation, cell–cell adhesion, defense, biofilm formation, motility, and cell-to-cell communication [1–3]. The bioactive metabolites secreted from the microbes exhibit a variety of biological activities, such as antibacterial, antifungal, antiviral, antiprotozoon, antimycoplasma, and antitumor activity [4, 5]. Such microbes have great application potential in pharAbbreviations: BA6B, Bacillus amyloliquefaciens 6B; LC–ESI–MS/MS, liquid chromatography coupled with electrospray ionization tandem mass spectrometry; β-OH FA, β-hydroxy fatty acid; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; NRPS, nonribosomal peptide synthetase. ∗ Address
for correspondence: Haresh Keharia PhD, BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, Vadtal Road, Sardar Patel University, Vallabh Vidyangar, Gujarat 388120, India. Tel.: +91 2692 234412; Fax: +91 2692 236475/237258; e-mail: [email protected]. Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 26 April 2013; accepted 14 October 2013 DOI: 10.1002/bab.1174 Published online in Wiley Online Library (wileyonlinelibrary.com)
maceutical industries for development of novel and more effective therapeutics, with low or no side effects. Although many synthetic or semisynthetic drugs have entered into the pharmaceutical industry, the natural products still occupy more than 50% of the pharmaceutical market [6]. After the advent of penicillin, most of the drug discovery programs in pharmaceutical industries were largely projected toward natural product screening and drug development [7–9]. In a natural product drug discovery program, screening, isolation, and characterization of natural bioactive products from the microbes are labor-intensive and exhaustive approaches accomplished with limited success over a long period of time. Tandem mass spectrometry is a rapid and sensitive approach for the screening and discovery of microbial natural products. The liquid chromatography coupled with the electrospray tandem mass spectrometry (LC–ESI–MS/MS) technique facilitates high-throughput screening and elucidation of structural information of biomolecules from a complex mixture [10–13]. The bacterium, identified as Bacillus amyloliquefaciens 6B (BA6B), was isolated from the Jakhao coast, India, and it exhibited a potent antifungal activity against Aspergillus niger, A. parasiticus, A. fumigatus, Fusarium oxysporum, Chrysosporium indicum, and Trichosporon sp. (unpublished data, 2012). The intact cell matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis revealed the
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FIG. 1
Primary structure of surfactin isomers produced by Bacillus strains.
production of surfactins by BA6B (Fig. S1 in the Supporting Information). The surfactins are lipopeptides with a heptacyclic lactone ring connected with β-hydroxy fatty acids (β-OH FA) (Fig. 1) [14–16]. This group of cyclic lipopeptides exhibits a wide range of variation in cyclic depsipeptide (Ile/Leu/Ile variations at positions 2, 4, and 7) as well as variations in the chain length of β-OH FA [14, 15]. Surfactins are known to exhibit a broad array of biological activities such as antifungal, antitumor, antiviral, and mosquitocidal and as a biosurfactant [17–20]. Surfactins have been shown to inhibit growth of Magnaporthe grisea, Curvularia lunata, Rhizoctonia bataticola, and F. oxysporum [21, 22]. Moreover, surfactins have been demonstrated to exhibit synergism with iturin and fengycins in their fungal antagonistic activity [21, 23]. The present study describes in detail the structural characterization of antifungal metabolites from a crude methanolic extract of bacterial isolate BA6B.
2. Materials and Methods 2.1. Culture and maintenance of bacterial strain The bacterium 6B was isolated from the soil of the Jakhao coast, India. The isolate 6B was identified as B. amyloliquefaciens 6B (BA6B) based on 16S rDNA analysis (Genebank accession no. JQ904625). The BA6B was maintained by subculturing on sterile Luria agar slopes at 30 ◦ C and stored at 4 ◦ C, as well as in the form of glycerol stocks at −20 ◦ C.
2.2. Production of antifungal compounds The preinoculum was prepared by an inoculating well-isolated single colony of BA6B into 50 mL of Luria broth (LB) (2%, w/v) in a 250-mL Erlenmeyer flask, which was incubated for 12 H on an orbital shaker (150 rpm) at 30 ◦ C. The preinoculum thus prepared was used for the production of antifungal compounds in LB medium. For production of antifungal metabolites, the inoculum was added to 500 mL LB in 1 L in the Erlenmeyer flask, to achieve an initial cell density of 0.05 absorbance units monitored using a visible spectrophotometer (Systronics, Ahmedabad, India) at 600 nm. The production medium after inoculation was incubated on an orbital shaker (150 rpm) at 30 ◦ C for 72 H.
2.3. Extraction of antifungal metabolites from fermentation broth The cells from the 72-H fermented broth of BA6B were removed by centrifugation at 10,000g at 4 ◦ C for 15 Min, and cell-free culture supernatant was collected in a separate sterile glass container and stored at 4 ◦ C until further processing. The antifungal compounds were precipitated by acidifying the
2
culture supernatant to pH 2 using 6 N HCl. The precipitated compounds were collected by centrifugation at 10,000g at 4 ◦ C for 25 min. The precipitates thus obtained were then solubilized in a minimum volume of HPLC-grade methanol (MeOH).
2.4. LC–ESI–MS/MS of antifungal extract from BA6B To characterize antifungal compounds from the methanolic antifungal extract of BA6B, LC–ESI–MS/MS approach was used as described previously by Pathak et al. [12] with minor modifications. In brief, 10 μL of antifungal methanolic extract (1 mg/mL) was run through a HPLC Zorbax 300 SB-column (Santa Clara, CA, USA) (4.6 × 150 mm2 , 5 μm particle size) at a 0.2 mL/Min flow rate using a MeOH/water/0.1% formic acid (FA) gradient (40–95% MeOH for 35 Min, 95% for 5 Min, and 95–50% for 5 Min) over a period of 45 Min. The antifungal compounds thus separated were analyzed online using a HCT ultra ETD II (Bruker Daltonics, Bremen, Germany) mass spectrometer. The separation profile was monitored using a UV detector at 226 nm and a total ion chromatogram. The mass spectra of separated compounds were recorded online over a range of m/z 300–2000 in a positive ion mode with a scan speed of 26,000 m/z Sec−1 . The spectra were averaged over four scans. The MS/MS spectra were acquired in auto MS/MS mode using He as a collision gas, and fragmentation amplitude (Vp-p) was kept at 1 for LC–MS/MS experiments. Data Analysis 4.0 software was used for LC–MS/MS data deconvolution (Bruker Daltonics).
3. Results and Discussion 3.1. Mass spectrometric characterization of antifungal extract of BA6B In an integrated mass spectrum (Fig. 2) of crude extract analyzed by LC–MS, nine different monoprotonated ions could be assigned with m/z varying from 994.7 to 1,096.8. Interestingly, in the mass spectrum, two series of ions could be identified. The ions within each series (series 1: m/z 994.7 [M1 ], 1,008.7 [M2 ], 1,022.7 [M3 ], 1,036.7 [M4 ], and 1,050.7 [M5 ]; series 2: m/z 1,054.7 [M6 ], 1,068.7 [M7 ], 1,082.7 [M8 ], and 1096.7 [M9 ]) varied in mass from each other by multiples of 14 Da. Furthermore, sodium adducts for all the monoprotonated ions described above except for m/z 994.7 could be distinctly assigned in the mass spectrum (Fig. 2). The presence of a cluster of ions differing in mass by multiples of 14 Da is a typical characteristic of cyclic lipopeptides, and based on the literature these molecular ions were putatively assigned as surfactins [12–15, 17, 24]. Moreover, these putatively assigned surfactins were found to be more hydrophobic as they eluted during a time period of 28–45 Min (92.4–95% MeOH, v/v) in the HPLC run. Several surfactins based on either variation in cyclic depsipeptide moiety or in the chain length of β-OH FA have been reported [12, 14, 15, 24]. The variations of Val/Ile/Leu
Novel Surfactins Produced by BA6B
FIG. 2
Integrated LC–ESI–MS spectrum of molecular mass ions separated in a reverse-phase HPLC Zorbax 300SB-column (4.6 × 150 mm2 , 5 μm particle size) at 0.2 μL/Min flow rate over a gradient of 45 Min.
at amino acid positions 2, 4, and 7 in the depsipeptide ring of surfactins have been documented in the literature (Fig. 1) [14, 15]. Along with these, the occurrence of Ala has also been reported at the second position in some surfactins [16]. The group of surfactin lipopeptides is known for their excellent biosurfactant property and other biological activities, such as hemolytic, antifungal, mosquitocidal, antiviral, and antitumor activities [15, 17–22]. To confirm the identity, each of these putatively assigned surfactins was further subjected to MS/MS analysis.
3.2. Characterization of surfactin microvariants 3.2.1. Isobaric cyclic lipodepsipeptide surfactins The LC–ESI–MS/MS spectra of protonated isobaric species at m/z 1,036.7 were found to elute at two different time intervals (Figs. 3 and 4) through a reverse-phase C18 column using a methanol gradient. In the MS/MS spectra of both of these species, b1 –b6 , b 1 -b 6 , y3 –y6 , and y 4 –y 6 type fragment ions could be assigned (Figs. 3 and 4). The precursor ion at m/z 1,036.7 eluting at a time interval of 44.0–44.4 Min represented two compounds (Fig. 3): b1 –b6 and y2 –y6 fragment ions represented cyclic lipodepsipeptide with a C14 β-OH FA-cyclo[E-I/L-I/L-I/L-I/L-D-I/L] sequence, whereas the b 1 – b 6 and y 3 –y 6 series of fragment ions represented another cyclic lipodepsipeptide with a C15 β-OH FA-cyclo[E-I/L-I/LV-I/L-D-I/L] sequence (Fig. 3). The cyclic lipodepsipeptide sequences with isobaric mass at m/z 1,036.7 could be identified as positional variants of C14 and C15 surfactins [15]. The MS/MS of monoprotonated molecular ions at m/z 1,036.7, which eluted after a retention time of 42.9–43.4 Min, was also found to consist of two coeluting isobaric molecules
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of different sequences (Fig. 4). The fragment ions at b1 –b6 and y2 –y6 in the MS/MS spectrum of m/z 1,036.7 represented the cyclic lipodepsipeptide with a C14 β-OH FA-cyclo [E-I/L-I/L-I/L-D-I/L-I/L] sequence, whereas another series of fragment ions b 1 –b 6 and y 3 –y 6 represented a C15 β-OH FAcyclo[E-I/L-I/L-V-D-I/L-I/L] cyclic lipodepsipeptide sequence. These isobaric molecules at m/z 1036, which eluted at two different time intervals from a C18 reverse-phase column, suggesting a difference in their polarity, exhibited variation at residues 5 and 6 of their peptide sequence (Figs. 3 and 4). Thus, these isobaric molecules could be identified as Asp positional variants of Ile2,4,7 and V4 , Ile2,7 surfactins. In this study, four positional variants of isobaric surfactins could be identified from the extract of BA6B. Apart from –b and –y type fragment ions, other ions at m/z 473.4, 572.4, 586.5, 685.5, and 699.5 shown in the MS/MS spectra could be assigned as the C-terminal fragments generated upon double hydrogen transfer during collision-induced dissociation (Figs. 3 and 4). The gain of 18 Da in the mass of these ions with respect to their corresponding –y ions may be attributed to the transfer of H+ and OH− ions to the C-terminal fragment ions during double hydrogen transfer as explained in detail by Yang et al. [25]. The C14 and C15 surfactin variants (Val/Ile/Leu4 , Asp6 ) were found to coelute and similarly C14 and C15 surfactin variants (Val/Ile/Leu4 , Asp5 ) also coeluted, suggesting that the position of Asp in surfactin isoforms has a greater influence on its retention in the C18 column than one carbon variation in the fatty acid chain length. As a consequence, C14 and C15 surfactin isoforms with Asp at the fifth position were eluted at different retention times (42.9–34.4 Min) (Figs. 4) than C14 and C15 surfactin isoforms with Asp at the sixth position (44.0–44.4 Min) (Fig. 3). The variation in retention times of isobaric surfactins with Asp5 , Ile/Leu6 , and Ile/Leu5 , Asp6 positional variants during reverse-phase HPLC may be attributed to the difference in their three-dimensional structure. Table 1 presents a list of surfactin isoforms identified from the methanolic extract of BA6B.
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FIG. 3
LC–ESI–MS/MS spectra of [M+H]+ ions at m/z 1,036.7 with retention time of 44.0–44.4 Min.
FIG. 4
LC–ESI–MS/MS spectra of [M+H]+ ion at m/z 1,036.7 with retention time of 42.9–43.4 Min.
Novel Surfactins Produced by BA6B
Surfactins identified from the methanolic extract of BA6B
TABLE 1 Number
Mass
Peptide sequence
Identification of surfactin
Reference
1
1,026.7
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C13 ) Surfactin (V4 ), [M + H]+
This study (new)
2
1,040.7
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C13 ) Surfactin (L/I4 ), [M + H]+
This study (new)
3
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C14 ) Surfactin (V4 ), [M + H]+
This study (new)
4
Linear β-OHFA-E-L/I-L/I-V-L/I-D-L/I
(C14 ) Surfactin (V4 ,D6 ), [M + H]+
This study (new)
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C14 ) Surfactin (L/I4 ), [M+H]+
This study (new)
6
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C15 ) Surfactin (V4 ), [M + H]+
This study (new)
7
Linear β-OHFA-E-L/I-L/I-L/I–L/I-L/I-D
(C14 ) Surfactin (L/I4 , D7 ), [M + H]+
This study (new)
8
Linear β-OHFA-E-L/I-L/I-V-L/I-L/I-D
(C15 ) Surfactin (V4 , D7 ), [M + H]+
This study (new)
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C15 ) Surfactin (L/I4 ), [M + H]+
This study (new)
10
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C16 ) Surfactin (V4 ), [M + H]+
This study (new)
11
Linear β-OHFA-E-L/I-L/I-L/I-L/I-D-L/I
(C15 ) Surfactin (V4 ,L/I5 ), [M + H]+
This study (new)
12
Linear β-OHFA-E-L/I-L/I-V-L/I-D-L/I
(C16 ) Surfactin (V4 ,L/I5 ), [M + H]+
This study (new)
13
1,082.7
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C16 ) Surfactin (L/I ), [M + H]
14
994.7
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C12 ) Surfactin (V4 ), [M + H]+
(15)
+
(15)
5
9
1,054.7
1,068.6
4
+
This study (new)
15
1,008.7
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C13 ) Surfactin (V ), [M + H]
16
1,022.7
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C13 ) Surfactin (L/I4 ), [M + H]+
17
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C14 ) Surfactin (V ), [M + H]
18
Cyclo(β-OHFA-E-L/I-L/I-L/I-L/I-D-L/I)
(C14 ) Surfactin (L/I4 ,D6 ), [M + H]+
Cyclo(β-OHFA-E-L/I-L/I-V-L/I-D-L/I)
(C15 ) Surfactin (V ,D ), [M + H]
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C14 ) Surfactin (L/I4 ), [M + H]+
21
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C15 ) Surfactin (V ), [M + H]
22
Cyclo(β-OHFA-E-L/I-L/I-L/I-L/I-D-L/I)
(C14 ) Surfactin (L/I4 ,D6 ), [M + H]+
Cyclo(β-OHFA-E-L/I-L/I-V-L/I-D-L/I)
(C15 ) Surfactin (V ,D ), [M + H]
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C14 ) Surfactin (L/I4 ), [M + H]+
19 20
1,036.7
23 24 25 26
1,050.7 1,064.7
4
4
4
6
4
4
This study
+
[15]
+
This study (new) [15]
+
6
This study (new)
[15]
+
This study (new) This study (new) This study
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C16 ) Surfactin (L/I ),[M + H]
+
This study
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C17 ) Surfactin (V4 ), [M + H]+
This study
3.2.2. Characterization of linear surfactins In addition to monoprotonated cyclic surfactin ions at m/z 1,008.7, 1,022.7, 1,036.7, 1,050.7, and 1,064.7 (Table 1), the corresponding protonated precursor ions with a gain in mass of 18 Da at m/z 1,026.7, 1,040.4, 1,054.7, 1,068.7, and 1,082.7 were also detected (Table 1 and Fig. S2 in the Supporting Information). These protonated ions seemed to be linear forms of corresponding cyclic surfactins and eluted over a retention time of 28–36 Min, that is, during a lower hydrophobic solvent fraction of gradient in comparison to cyclic surfactins (35–
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45 Min retention time). The linear surfactins have a free β-hydroxyl group and a free carboxyl group and as a result are more polar and thus elute earlier in comparison to their corresponding cyclic forms from a C-18 reverse-phase column. To further confirm the identity, each of these putative linear surfactin ions was subjected to tandem mass spectrometric characterization. Figures 5 and 6 present the MS/MS spectra of two isobaric molecules at m/z 1,054.7 eluting at two different time intervals: (1) 32.1–32.6 and (2) 34.9–35.3 Min, respectively. The fragment
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FIG. 5
LC–ESI–MS/MS spectra of [M+H]+ ion at m/z 1,054.7 with retention time of 32.1–32.6 Min.
ions b1 –b6 and y4 –y6 in the MS/MS spectrum of ion at m/z 1,054.7 (retention time 32.1–32.6 Min) corresponded to a linear surfactin sequence, C14 β-OH FA-E-I/L-I/L-I/L-D-I/L-I/ L-COOH (Fig. 5). In the same spectrum, another set of fragment ions b 1 –b 6 and y 4 –y 6 enabled the assignment of C15 β-OH FA-E-I/L-I/L-I/L-V-D-I/L-I/L-COOH linear surfactin (Fig. 5). In the case of cyclic surfactins, b6 ions in the MS/MS spectra represented loss of residual mass (113 Da) corresponding to the Ile/Leu residue (Figs. 3 and 4). In Figure 5, b6 and b 6 ions at m/z 923.6 could be observed upon loss of 131.1 Da corresponding to the loss of Ile/Leu-COOH from a surfactin molecular ion. The evidence of loss of amino acids with free C-terminal –COOH further supported the presence of the linear surfactin ion with m/z at 1,054.7. Figure 6 shows the MS/MS spectrum of a mixture of two molecules with the same molecular mass ion at m/z 1,054.7. From the b1 –b6 and y5 –y6 fragment ions, C14 β-OH FA-E-I/L-I/LI/L-I/L-I/L-D-COOH could be assigned, whereas the fragment ions b 1 –b 6 and y 4 , y 6 enabled the assignment of C15 β-OH FAE-I/L-I/L-V-I/L-I/L-D-COOH lipopeptide sequence. The assigned sequences are similar to C14 and C15 linear surfactin isoforms with the replacement of Asp5 to Ile/Leu5 and Ile/Leu7 to Asp7 . Other ions at m/z 1,068.7, 1,076.7, 1,082.7, 1,090.7, 1,096.7, and 1,104.4 were also confirmed as linear surfactins. The linear surfactin ions identified in this study are summarized in Table 1. The methanolic extract of BA6B was composed of both cyclic and linear surfactins. However, the amount of linear surfactins was too low in comparison to cyclic surfactins.
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Although methanolic extract was composed of a similar number of both the cyclic and linear surfactin species, the amount of linear surfactins corresponded to only 10% of the total surfactin amount. The linear surfactins in the methonolic extract of BA6B might have resulted either from the cleavage of the depsipeptide ring during downstream processing from the fermentation broth or more likely the noncyclized intermediates of a surfactin biosynthetic pathway [26–28]. The surfactins are nonribosomally synthesized peptides and require nonribosomal peptide synthetases (NRPS) for their biosynthesis [26–28]. The NRPS are multimodular megadalton enzyme complexes wherein each module acts as an enzyme. Each module comprises mainly three domains, adenylation (A), thiolation (T), and condensation (C). The “A” domain selects amino acid and activates the amino acid, the “T” domain loads this activated amino acid and transfers to the “C” domain, whereas the “C” domain catalyzes the formation of the amide bond [28]. The srfA operon in B. subtilis contains four open reading frames, srfAA, srfAB, srfAC, and srfAD, which encode the surfactin synthetase multienzyme system [22]. The srfAA-srfAC encodes the first seven modules of the surfactin synthetase, each corresponding to a respective amino acid in surfactin heptapeptide [26–28]. Some of the modules in the NRPS system may exhibit reduced specificity toward structurally similar amino acids, resulting in the generation of microheterogeneity in peptides [29]. The fourth open reading frame (ORF), srfAD, encodes for the thioesterase domain (TE domain), responsible for cyclization of linear surfactin. The production of surfactins by B. amyloliquefaciens strains as well as the presence of complete srfA operon in the genome of plant growth promoting soil bacterium, B. amyloliquefaciens FZB42 has been well documented [22, 23, 30]. The structural surfactin
Novel Surfactins Produced by BA6B
FIG. 6
LC–ESI–MS/MS spectra of [M+H]+ ion at m/z 1,054.7 with retention time of 34.9–35.3 Min.
microvariants identified in this study may be attributed either to reduced substrate selectivity or to the difference in the organization of modules of surfactin synthetase machinery. This study presents the structural characterization of surfactin variants from the methanolic extract of fungal antagonist strain B. amyloliquefaciens 6B using a LC–ESI–MS2 technique. A total of 26 surfactin species belonging to 11 unique molecular mass ions could be identified (Table 1). Among the 11 unique molecular mass ions, six mass ions at m/z 994.7, 1,008.7, 1,022.7, 1,036.7, 1,050.7, and 1,064.7 corresponded to cyclic species, whereas the rest of the mass ions were identified as linear surfactins (m/z at 1,026.7, 1,040.7, 1,054.7, 1,068.6, and 1,082.7). Each unique molecular mass ion at m/z 1,022.7, 1,036.7, 1,040.7, 1,054.7, 1,068.6, and 1,064.7 represented two to four isobaric species of surfactins. To the best of our knowledge, this is the first report identifying maximum numbers of variants of surfactins synthesized by B. amyloliquefaciens 6B.
4. Acknowledgement K.P. acknowledges Professor Balaram for giving access to the mass spectrometer facility and teaching mass spectrometry. The authors declare no conflict of interest.
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5. References [1] Gram, L., de Nys, R., Maximilien, R., Givskov, M., Steinberg, P., and Kjelleberg, S. (1996) Appl. Env. Microbiol. 62, 4284–4287. [2] Visick, K. L., and Fuqua, C. (2005) J. Bacteriol. 187, 5507–5519. [3] Hayes, C. S., and Low, D. A. (2009) Curr. Opin. Microbiol. 6, 667–673. [4] Knight, V., Sanglier, J. J., DiTullio, D., Braccili, S., Bonner, P., Waters, J., Hughes, D., and Zhang, L. (2003) Appl. Microbiol. Biotechnol. 62, 446–458. ´ [5] Vicente, M. F., Basilio, A., Cabello, A., and Pelaez, F. (2003) Clin. Microbiol. Infect. 9, 15–32. [6] Harvey, A. L. (2008) Drug Discov. Today 19, 894–901. [7] Harvey, A. L. (2000) Drug Discov. Today 7, 294–300. [8] Newman, D. J., and Cragg, G. M. (2007) J. Nat. Prod. 70, 461–477. [9] Rishton, G. M. (2008) Am. J. Cardiol. 101, 43–49. [10] Bodnar, W. M., Blackburn, R. K., Krise, J. M., and Moseley, M. A. (2003) J. Am. Soc. Mass Spectrom. 14, 971–979. [11] Pecci, Y., Rivardo, F., Martinotti, M. G., and Allegrone, G. (2010) J. Mass. Spectrom. 45, 772–778. [12] Pathak, K. V., Keharia, H., Gupta, K., Thakur, S. S., and Balaram, P. (2012) J. Am. Soc. Mass Spectrom. 10, 1716–1728. [13] Pinkse, M. W., Uitto, P. M., Hilhorst, M. J., Ooms, B., and Heck, A. J. (2004) Anal. Chem. 76, 3935–3943. [14] Peypoux, F., Bonmatin, J. M., and Wallach, J. (1999) Appl. Microbiol. Biotechnol. 51, 553–563. [15] Kowall, M., Vater, J., Kluge, B., Stein, T., Franke, P., and Ziessow, D. (1998) J. Colloid Interface Sci. 204, 1–8. [16] Peypoux, F., Bonmatin, J. M., Labbe, H., Grangemard, I., Das, B. C., Ptak, M., Wallach, J., and Michel, G. (1994) Eur. J. Biochem. 224, 89–96. [17] Pathak, K. V., and Keharia, H. (2013) 3 Biotech. doi. 10.1007/s13205-013-0119-3. [18] Kracht, M., Rokos, H., Ozel, M., Kowall, M., Pauli, G., and Vater, J. (1999) J. Antibiot. 52, 613–619. [19] Kim, S. Y., Kim, J. M., Kim, S. H., Bae, H. J., Yi, H., and Yoon, S. H. (2007) FEBS Lett. 581, 865–871.
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Biotechnology and Applied Biochemistry [20] Geetha, L., Manonmani, A. M., and Paily, K. P. (2010) Appl. Microbiol. Biotechnol. 86, 1737–1744. [21] Tendulkar, S. R., Saikumari, Y. K., Patel, V., Raghotoma, S., Munshi, T. K., Balaram, P., and Chatoo, B. (2007) J. Appl. Microbiol. 103, 2331– 2339. [22] Vitullo, D., Di Pietro, A., Romano, A., Lanzotti, V., and Lima, G. (2012) Plant Path. 61, 689–699. [23] Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B., and Fickers, P. (2009) Microb. Cell Fact. 8, 63– 75. [24] Madonna, A. J., Voorhees, K. J., Taranenko, N. I., Laiko, V. V., and Doroshenko, V. M. (2003) Anal. Chem. 75, 1628–1637.
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[25] Yang, S. Z., Wei, D. Z., and Mu, B. Z. (2006) J. Biochem. Biophys. Methods 168, 69–74. [26] Marahiel, M. A., Stachelhaus, T., and Mootz, H. D. (1997) Chem. Rev. 97, 2651–2674. [27] Vollenbroich, D., Mehta, N., Zuber, P., Vater, J., and Kamp, R. M. (1994) J. Bacteriol. 176, 395–400. [28] Menkhaus, M., Ullrich, C., Kluge, B., Vater, J., Vollenbroich, D., and Kamp, R. M. (1993) J. Biol. Chem. 268, 7678–7684. [29] Shu, H. Y., Lin, G. H., Wu, Y. C., Tschen, J. S., Liu, S. T. (2002) Biochem. Biophys. Res. Commun. 292, 789–793. [30] Koumoutsi, A., Chen, X. H., Henne, A., Liesegang, H., Hitzeroth, G., Franke, P., Vater, J., and Borriss, R. (2004) J. Bacteriol. 186, 1084–1096.
Novel Surfactins Produced by BA6B
Khyati V. Pathak Anjali Bose Haresh Keharia∗
BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, Sardar Patel University, Vallabh Vidyangar, Gujarat, India
Abstract The broad-spectrum fungal antagonist, Bacillus amyloliquefaciens 6B (BA6B), isolated from the Jakhao coast of Kutch, India, was investigated for its antifungal metabolites using mass spectrometry. The cyclic lipopeptides harvested from the cell-free fermentation broth of BA6B by acid precipitation and subsequently dissolved in methanol were subjected to liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI–MS/MS) for their identification and sequence determination. The 26 types of surfactin variants were identified from the methanolic extract
by LC–ESI–MS/MS analysis. Among 26 surfactin species, several new cyclic as well as acyclic surfactin variants based on the variation in the β-hydroxy fatty acid (β-OH FA) chain length and/or in amino acid positions 4, 5, 6, and 7 were identified. The mass spectrometric analysis of crude extract also enabled the identification of 11 unique molecular mass ions with minimum two or maximum four types of isobaric peptide variants. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 0, Number 0, Pages 1–8, 2014
Keywords: Bacillus amyloliquefaciens, surfactin, isobaric cyclic lipopeptides, mass spectrometry
1. Introduction Microbial cells are one of the most elegant chemical factories manufacturing a plethora of bioactive natural compounds to perform various physiological functions, such as growth and its regulation, cell–cell adhesion, defense, biofilm formation, motility, and cell-to-cell communication [1–3]. The bioactive metabolites secreted from the microbes exhibit a variety of biological activities, such as antibacterial, antifungal, antiviral, antiprotozoon, antimycoplasma, and antitumor activity [4, 5]. Such microbes have great application potential in pharAbbreviations: BA6B, Bacillus amyloliquefaciens 6B; LC–ESI–MS/MS, liquid chromatography coupled with electrospray ionization tandem mass spectrometry; β-OH FA, β-hydroxy fatty acid; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; NRPS, nonribosomal peptide synthetase. ∗ Address
for correspondence: Haresh Keharia PhD, BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, Vadtal Road, Sardar Patel University, Vallabh Vidyangar, Gujarat 388120, India. Tel.: +91 2692 234412; Fax: +91 2692 236475/237258; e-mail: [email protected]. Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 26 April 2013; accepted 14 October 2013 DOI: 10.1002/bab.1174 Published online in Wiley Online Library (wileyonlinelibrary.com)
maceutical industries for development of novel and more effective therapeutics, with low or no side effects. Although many synthetic or semisynthetic drugs have entered into the pharmaceutical industry, the natural products still occupy more than 50% of the pharmaceutical market [6]. After the advent of penicillin, most of the drug discovery programs in pharmaceutical industries were largely projected toward natural product screening and drug development [7–9]. In a natural product drug discovery program, screening, isolation, and characterization of natural bioactive products from the microbes are labor-intensive and exhaustive approaches accomplished with limited success over a long period of time. Tandem mass spectrometry is a rapid and sensitive approach for the screening and discovery of microbial natural products. The liquid chromatography coupled with the electrospray tandem mass spectrometry (LC–ESI–MS/MS) technique facilitates high-throughput screening and elucidation of structural information of biomolecules from a complex mixture [10–13]. The bacterium, identified as Bacillus amyloliquefaciens 6B (BA6B), was isolated from the Jakhao coast, India, and it exhibited a potent antifungal activity against Aspergillus niger, A. parasiticus, A. fumigatus, Fusarium oxysporum, Chrysosporium indicum, and Trichosporon sp. (unpublished data, 2012). The intact cell matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis revealed the
1
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FIG. 1
Primary structure of surfactin isomers produced by Bacillus strains.
production of surfactins by BA6B (Fig. S1 in the Supporting Information). The surfactins are lipopeptides with a heptacyclic lactone ring connected with β-hydroxy fatty acids (β-OH FA) (Fig. 1) [14–16]. This group of cyclic lipopeptides exhibits a wide range of variation in cyclic depsipeptide (Ile/Leu/Ile variations at positions 2, 4, and 7) as well as variations in the chain length of β-OH FA [14, 15]. Surfactins are known to exhibit a broad array of biological activities such as antifungal, antitumor, antiviral, and mosquitocidal and as a biosurfactant [17–20]. Surfactins have been shown to inhibit growth of Magnaporthe grisea, Curvularia lunata, Rhizoctonia bataticola, and F. oxysporum [21, 22]. Moreover, surfactins have been demonstrated to exhibit synergism with iturin and fengycins in their fungal antagonistic activity [21, 23]. The present study describes in detail the structural characterization of antifungal metabolites from a crude methanolic extract of bacterial isolate BA6B.
2. Materials and Methods 2.1. Culture and maintenance of bacterial strain The bacterium 6B was isolated from the soil of the Jakhao coast, India. The isolate 6B was identified as B. amyloliquefaciens 6B (BA6B) based on 16S rDNA analysis (Genebank accession no. JQ904625). The BA6B was maintained by subculturing on sterile Luria agar slopes at 30 ◦ C and stored at 4 ◦ C, as well as in the form of glycerol stocks at −20 ◦ C.
2.2. Production of antifungal compounds The preinoculum was prepared by an inoculating well-isolated single colony of BA6B into 50 mL of Luria broth (LB) (2%, w/v) in a 250-mL Erlenmeyer flask, which was incubated for 12 H on an orbital shaker (150 rpm) at 30 ◦ C. The preinoculum thus prepared was used for the production of antifungal compounds in LB medium. For production of antifungal metabolites, the inoculum was added to 500 mL LB in 1 L in the Erlenmeyer flask, to achieve an initial cell density of 0.05 absorbance units monitored using a visible spectrophotometer (Systronics, Ahmedabad, India) at 600 nm. The production medium after inoculation was incubated on an orbital shaker (150 rpm) at 30 ◦ C for 72 H.
2.3. Extraction of antifungal metabolites from fermentation broth The cells from the 72-H fermented broth of BA6B were removed by centrifugation at 10,000g at 4 ◦ C for 15 Min, and cell-free culture supernatant was collected in a separate sterile glass container and stored at 4 ◦ C until further processing. The antifungal compounds were precipitated by acidifying the
2
culture supernatant to pH 2 using 6 N HCl. The precipitated compounds were collected by centrifugation at 10,000g at 4 ◦ C for 25 min. The precipitates thus obtained were then solubilized in a minimum volume of HPLC-grade methanol (MeOH).
2.4. LC–ESI–MS/MS of antifungal extract from BA6B To characterize antifungal compounds from the methanolic antifungal extract of BA6B, LC–ESI–MS/MS approach was used as described previously by Pathak et al. [12] with minor modifications. In brief, 10 μL of antifungal methanolic extract (1 mg/mL) was run through a HPLC Zorbax 300 SB-column (Santa Clara, CA, USA) (4.6 × 150 mm2 , 5 μm particle size) at a 0.2 mL/Min flow rate using a MeOH/water/0.1% formic acid (FA) gradient (40–95% MeOH for 35 Min, 95% for 5 Min, and 95–50% for 5 Min) over a period of 45 Min. The antifungal compounds thus separated were analyzed online using a HCT ultra ETD II (Bruker Daltonics, Bremen, Germany) mass spectrometer. The separation profile was monitored using a UV detector at 226 nm and a total ion chromatogram. The mass spectra of separated compounds were recorded online over a range of m/z 300–2000 in a positive ion mode with a scan speed of 26,000 m/z Sec−1 . The spectra were averaged over four scans. The MS/MS spectra were acquired in auto MS/MS mode using He as a collision gas, and fragmentation amplitude (Vp-p) was kept at 1 for LC–MS/MS experiments. Data Analysis 4.0 software was used for LC–MS/MS data deconvolution (Bruker Daltonics).
3. Results and Discussion 3.1. Mass spectrometric characterization of antifungal extract of BA6B In an integrated mass spectrum (Fig. 2) of crude extract analyzed by LC–MS, nine different monoprotonated ions could be assigned with m/z varying from 994.7 to 1,096.8. Interestingly, in the mass spectrum, two series of ions could be identified. The ions within each series (series 1: m/z 994.7 [M1 ], 1,008.7 [M2 ], 1,022.7 [M3 ], 1,036.7 [M4 ], and 1,050.7 [M5 ]; series 2: m/z 1,054.7 [M6 ], 1,068.7 [M7 ], 1,082.7 [M8 ], and 1096.7 [M9 ]) varied in mass from each other by multiples of 14 Da. Furthermore, sodium adducts for all the monoprotonated ions described above except for m/z 994.7 could be distinctly assigned in the mass spectrum (Fig. 2). The presence of a cluster of ions differing in mass by multiples of 14 Da is a typical characteristic of cyclic lipopeptides, and based on the literature these molecular ions were putatively assigned as surfactins [12–15, 17, 24]. Moreover, these putatively assigned surfactins were found to be more hydrophobic as they eluted during a time period of 28–45 Min (92.4–95% MeOH, v/v) in the HPLC run. Several surfactins based on either variation in cyclic depsipeptide moiety or in the chain length of β-OH FA have been reported [12, 14, 15, 24]. The variations of Val/Ile/Leu
Novel Surfactins Produced by BA6B
FIG. 2
Integrated LC–ESI–MS spectrum of molecular mass ions separated in a reverse-phase HPLC Zorbax 300SB-column (4.6 × 150 mm2 , 5 μm particle size) at 0.2 μL/Min flow rate over a gradient of 45 Min.
at amino acid positions 2, 4, and 7 in the depsipeptide ring of surfactins have been documented in the literature (Fig. 1) [14, 15]. Along with these, the occurrence of Ala has also been reported at the second position in some surfactins [16]. The group of surfactin lipopeptides is known for their excellent biosurfactant property and other biological activities, such as hemolytic, antifungal, mosquitocidal, antiviral, and antitumor activities [15, 17–22]. To confirm the identity, each of these putatively assigned surfactins was further subjected to MS/MS analysis.
3.2. Characterization of surfactin microvariants 3.2.1. Isobaric cyclic lipodepsipeptide surfactins The LC–ESI–MS/MS spectra of protonated isobaric species at m/z 1,036.7 were found to elute at two different time intervals (Figs. 3 and 4) through a reverse-phase C18 column using a methanol gradient. In the MS/MS spectra of both of these species, b1 –b6 , b 1 -b 6 , y3 –y6 , and y 4 –y 6 type fragment ions could be assigned (Figs. 3 and 4). The precursor ion at m/z 1,036.7 eluting at a time interval of 44.0–44.4 Min represented two compounds (Fig. 3): b1 –b6 and y2 –y6 fragment ions represented cyclic lipodepsipeptide with a C14 β-OH FA-cyclo[E-I/L-I/L-I/L-I/L-D-I/L] sequence, whereas the b 1 – b 6 and y 3 –y 6 series of fragment ions represented another cyclic lipodepsipeptide with a C15 β-OH FA-cyclo[E-I/L-I/LV-I/L-D-I/L] sequence (Fig. 3). The cyclic lipodepsipeptide sequences with isobaric mass at m/z 1,036.7 could be identified as positional variants of C14 and C15 surfactins [15]. The MS/MS of monoprotonated molecular ions at m/z 1,036.7, which eluted after a retention time of 42.9–43.4 Min, was also found to consist of two coeluting isobaric molecules
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of different sequences (Fig. 4). The fragment ions at b1 –b6 and y2 –y6 in the MS/MS spectrum of m/z 1,036.7 represented the cyclic lipodepsipeptide with a C14 β-OH FA-cyclo [E-I/L-I/L-I/L-D-I/L-I/L] sequence, whereas another series of fragment ions b 1 –b 6 and y 3 –y 6 represented a C15 β-OH FAcyclo[E-I/L-I/L-V-D-I/L-I/L] cyclic lipodepsipeptide sequence. These isobaric molecules at m/z 1036, which eluted at two different time intervals from a C18 reverse-phase column, suggesting a difference in their polarity, exhibited variation at residues 5 and 6 of their peptide sequence (Figs. 3 and 4). Thus, these isobaric molecules could be identified as Asp positional variants of Ile2,4,7 and V4 , Ile2,7 surfactins. In this study, four positional variants of isobaric surfactins could be identified from the extract of BA6B. Apart from –b and –y type fragment ions, other ions at m/z 473.4, 572.4, 586.5, 685.5, and 699.5 shown in the MS/MS spectra could be assigned as the C-terminal fragments generated upon double hydrogen transfer during collision-induced dissociation (Figs. 3 and 4). The gain of 18 Da in the mass of these ions with respect to their corresponding –y ions may be attributed to the transfer of H+ and OH− ions to the C-terminal fragment ions during double hydrogen transfer as explained in detail by Yang et al. [25]. The C14 and C15 surfactin variants (Val/Ile/Leu4 , Asp6 ) were found to coelute and similarly C14 and C15 surfactin variants (Val/Ile/Leu4 , Asp5 ) also coeluted, suggesting that the position of Asp in surfactin isoforms has a greater influence on its retention in the C18 column than one carbon variation in the fatty acid chain length. As a consequence, C14 and C15 surfactin isoforms with Asp at the fifth position were eluted at different retention times (42.9–34.4 Min) (Figs. 4) than C14 and C15 surfactin isoforms with Asp at the sixth position (44.0–44.4 Min) (Fig. 3). The variation in retention times of isobaric surfactins with Asp5 , Ile/Leu6 , and Ile/Leu5 , Asp6 positional variants during reverse-phase HPLC may be attributed to the difference in their three-dimensional structure. Table 1 presents a list of surfactin isoforms identified from the methanolic extract of BA6B.
3
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FIG. 3
LC–ESI–MS/MS spectra of [M+H]+ ions at m/z 1,036.7 with retention time of 44.0–44.4 Min.
FIG. 4
LC–ESI–MS/MS spectra of [M+H]+ ion at m/z 1,036.7 with retention time of 42.9–43.4 Min.
Novel Surfactins Produced by BA6B
Surfactins identified from the methanolic extract of BA6B
TABLE 1 Number
Mass
Peptide sequence
Identification of surfactin
Reference
1
1,026.7
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C13 ) Surfactin (V4 ), [M + H]+
This study (new)
2
1,040.7
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C13 ) Surfactin (L/I4 ), [M + H]+
This study (new)
3
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C14 ) Surfactin (V4 ), [M + H]+
This study (new)
4
Linear β-OHFA-E-L/I-L/I-V-L/I-D-L/I
(C14 ) Surfactin (V4 ,D6 ), [M + H]+
This study (new)
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C14 ) Surfactin (L/I4 ), [M+H]+
This study (new)
6
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C15 ) Surfactin (V4 ), [M + H]+
This study (new)
7
Linear β-OHFA-E-L/I-L/I-L/I–L/I-L/I-D
(C14 ) Surfactin (L/I4 , D7 ), [M + H]+
This study (new)
8
Linear β-OHFA-E-L/I-L/I-V-L/I-L/I-D
(C15 ) Surfactin (V4 , D7 ), [M + H]+
This study (new)
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C15 ) Surfactin (L/I4 ), [M + H]+
This study (new)
10
Linear β-OHFA-E-L/I-L/I-V-D-L/I-L/I
(C16 ) Surfactin (V4 ), [M + H]+
This study (new)
11
Linear β-OHFA-E-L/I-L/I-L/I-L/I-D-L/I
(C15 ) Surfactin (V4 ,L/I5 ), [M + H]+
This study (new)
12
Linear β-OHFA-E-L/I-L/I-V-L/I-D-L/I
(C16 ) Surfactin (V4 ,L/I5 ), [M + H]+
This study (new)
13
1,082.7
Linear β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I
(C16 ) Surfactin (L/I ), [M + H]
14
994.7
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C12 ) Surfactin (V4 ), [M + H]+
(15)
+
(15)
5
9
1,054.7
1,068.6
4
+
This study (new)
15
1,008.7
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C13 ) Surfactin (V ), [M + H]
16
1,022.7
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C13 ) Surfactin (L/I4 ), [M + H]+
17
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C14 ) Surfactin (V ), [M + H]
18
Cyclo(β-OHFA-E-L/I-L/I-L/I-L/I-D-L/I)
(C14 ) Surfactin (L/I4 ,D6 ), [M + H]+
Cyclo(β-OHFA-E-L/I-L/I-V-L/I-D-L/I)
(C15 ) Surfactin (V ,D ), [M + H]
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C14 ) Surfactin (L/I4 ), [M + H]+
21
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C15 ) Surfactin (V ), [M + H]
22
Cyclo(β-OHFA-E-L/I-L/I-L/I-L/I-D-L/I)
(C14 ) Surfactin (L/I4 ,D6 ), [M + H]+
Cyclo(β-OHFA-E-L/I-L/I-V-L/I-D-L/I)
(C15 ) Surfactin (V ,D ), [M + H]
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C14 ) Surfactin (L/I4 ), [M + H]+
19 20
1,036.7
23 24 25 26
1,050.7 1,064.7
4
4
4
6
4
4
This study
+
[15]
+
This study (new) [15]
+
6
This study (new)
[15]
+
This study (new) This study (new) This study
Cyclo(β-OHFA-E-L/I-L/I-L/I-D-L/I-L/I)
(C16 ) Surfactin (L/I ),[M + H]
+
This study
Cyclo(β-OHFA-E-L/I-L/I-V-D-L/I-L/I)
(C17 ) Surfactin (V4 ), [M + H]+
This study
3.2.2. Characterization of linear surfactins In addition to monoprotonated cyclic surfactin ions at m/z 1,008.7, 1,022.7, 1,036.7, 1,050.7, and 1,064.7 (Table 1), the corresponding protonated precursor ions with a gain in mass of 18 Da at m/z 1,026.7, 1,040.4, 1,054.7, 1,068.7, and 1,082.7 were also detected (Table 1 and Fig. S2 in the Supporting Information). These protonated ions seemed to be linear forms of corresponding cyclic surfactins and eluted over a retention time of 28–36 Min, that is, during a lower hydrophobic solvent fraction of gradient in comparison to cyclic surfactins (35–
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4
45 Min retention time). The linear surfactins have a free β-hydroxyl group and a free carboxyl group and as a result are more polar and thus elute earlier in comparison to their corresponding cyclic forms from a C-18 reverse-phase column. To further confirm the identity, each of these putative linear surfactin ions was subjected to tandem mass spectrometric characterization. Figures 5 and 6 present the MS/MS spectra of two isobaric molecules at m/z 1,054.7 eluting at two different time intervals: (1) 32.1–32.6 and (2) 34.9–35.3 Min, respectively. The fragment
5
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FIG. 5
LC–ESI–MS/MS spectra of [M+H]+ ion at m/z 1,054.7 with retention time of 32.1–32.6 Min.
ions b1 –b6 and y4 –y6 in the MS/MS spectrum of ion at m/z 1,054.7 (retention time 32.1–32.6 Min) corresponded to a linear surfactin sequence, C14 β-OH FA-E-I/L-I/L-I/L-D-I/L-I/ L-COOH (Fig. 5). In the same spectrum, another set of fragment ions b 1 –b 6 and y 4 –y 6 enabled the assignment of C15 β-OH FA-E-I/L-I/L-I/L-V-D-I/L-I/L-COOH linear surfactin (Fig. 5). In the case of cyclic surfactins, b6 ions in the MS/MS spectra represented loss of residual mass (113 Da) corresponding to the Ile/Leu residue (Figs. 3 and 4). In Figure 5, b6 and b 6 ions at m/z 923.6 could be observed upon loss of 131.1 Da corresponding to the loss of Ile/Leu-COOH from a surfactin molecular ion. The evidence of loss of amino acids with free C-terminal –COOH further supported the presence of the linear surfactin ion with m/z at 1,054.7. Figure 6 shows the MS/MS spectrum of a mixture of two molecules with the same molecular mass ion at m/z 1,054.7. From the b1 –b6 and y5 –y6 fragment ions, C14 β-OH FA-E-I/L-I/LI/L-I/L-I/L-D-COOH could be assigned, whereas the fragment ions b 1 –b 6 and y 4 , y 6 enabled the assignment of C15 β-OH FAE-I/L-I/L-V-I/L-I/L-D-COOH lipopeptide sequence. The assigned sequences are similar to C14 and C15 linear surfactin isoforms with the replacement of Asp5 to Ile/Leu5 and Ile/Leu7 to Asp7 . Other ions at m/z 1,068.7, 1,076.7, 1,082.7, 1,090.7, 1,096.7, and 1,104.4 were also confirmed as linear surfactins. The linear surfactin ions identified in this study are summarized in Table 1. The methanolic extract of BA6B was composed of both cyclic and linear surfactins. However, the amount of linear surfactins was too low in comparison to cyclic surfactins.
6
Although methanolic extract was composed of a similar number of both the cyclic and linear surfactin species, the amount of linear surfactins corresponded to only 10% of the total surfactin amount. The linear surfactins in the methonolic extract of BA6B might have resulted either from the cleavage of the depsipeptide ring during downstream processing from the fermentation broth or more likely the noncyclized intermediates of a surfactin biosynthetic pathway [26–28]. The surfactins are nonribosomally synthesized peptides and require nonribosomal peptide synthetases (NRPS) for their biosynthesis [26–28]. The NRPS are multimodular megadalton enzyme complexes wherein each module acts as an enzyme. Each module comprises mainly three domains, adenylation (A), thiolation (T), and condensation (C). The “A” domain selects amino acid and activates the amino acid, the “T” domain loads this activated amino acid and transfers to the “C” domain, whereas the “C” domain catalyzes the formation of the amide bond [28]. The srfA operon in B. subtilis contains four open reading frames, srfAA, srfAB, srfAC, and srfAD, which encode the surfactin synthetase multienzyme system [22]. The srfAA-srfAC encodes the first seven modules of the surfactin synthetase, each corresponding to a respective amino acid in surfactin heptapeptide [26–28]. Some of the modules in the NRPS system may exhibit reduced specificity toward structurally similar amino acids, resulting in the generation of microheterogeneity in peptides [29]. The fourth open reading frame (ORF), srfAD, encodes for the thioesterase domain (TE domain), responsible for cyclization of linear surfactin. The production of surfactins by B. amyloliquefaciens strains as well as the presence of complete srfA operon in the genome of plant growth promoting soil bacterium, B. amyloliquefaciens FZB42 has been well documented [22, 23, 30]. The structural surfactin
Novel Surfactins Produced by BA6B
FIG. 6
LC–ESI–MS/MS spectra of [M+H]+ ion at m/z 1,054.7 with retention time of 34.9–35.3 Min.
microvariants identified in this study may be attributed either to reduced substrate selectivity or to the difference in the organization of modules of surfactin synthetase machinery. This study presents the structural characterization of surfactin variants from the methanolic extract of fungal antagonist strain B. amyloliquefaciens 6B using a LC–ESI–MS2 technique. A total of 26 surfactin species belonging to 11 unique molecular mass ions could be identified (Table 1). Among the 11 unique molecular mass ions, six mass ions at m/z 994.7, 1,008.7, 1,022.7, 1,036.7, 1,050.7, and 1,064.7 corresponded to cyclic species, whereas the rest of the mass ions were identified as linear surfactins (m/z at 1,026.7, 1,040.7, 1,054.7, 1,068.6, and 1,082.7). Each unique molecular mass ion at m/z 1,022.7, 1,036.7, 1,040.7, 1,054.7, 1,068.6, and 1,064.7 represented two to four isobaric species of surfactins. To the best of our knowledge, this is the first report identifying maximum numbers of variants of surfactins synthesized by B. amyloliquefaciens 6B.
4. Acknowledgement K.P. acknowledges Professor Balaram for giving access to the mass spectrometer facility and teaching mass spectrometry. The authors declare no conflict of interest.
Biotechnology and Applied Biochemistry
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Novel Surfactins Produced by BA6B
