Abstract
The banyan endophyte, Bacillus subtilis K1, produces a complex mixture of lipopeptides exhibiting potent antifungal activity. These lipopeptides were purified by high-performance liquid chromatography and analyzed using MALDI-TOF-MS as well as liquid chromatography coupled with ESI-MS. A heterogenous mixture of lipopeptides belonging to three different families of cyclic lipopeptides, viz., fengycins, iturins and surfactins, was detected in the cell-free extracellular extract of B. subtilis K1. The detailed mass spectrometric characterization revealed the presence of four variants of iturin A and three variants of iturin C varying in the β-amino fatty acid chain length from C13 to C17. The MS/MS of monovalent alkali metal ion adducts (Na and K) of iturin suggested the Glu4 as a binding site for metal ion. The LC-ESI-MS/MS analysis of surfactins enabled the identification of seven surfactin variants with the variations in Val/Ile/Leu at position 4 and C13–C17 β-hydroxy fatty acids. This study demonstrates the application of tandem mass spectrometry in identification of closely related lipopeptides from a heterogenous mixture obtained from a natural source. Furthermore, this is the first report of an endophytic bacillus strain co-producing so many variants of surfactins and iturins.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-013-0151-3) contains supplementary material, which is available to authorized users.
Highlights
Endophytes are microorganisms which reside within plant tissues without causing any harm to host and they represent potential source of novel bioactive metabolites (Strobel 2006)
Iturins are broad spectrum potent antifungal heptapeptides cyclized by amide bond formed between a-COO group of seventh amino acid and b-NH2 group of b-amino fatty acid, which in turn is peptide bonded through its a-COO group to N-terminal amino acid
The MALDI-TOF MS analysis of HPLC purified fractions of crude antifungal extract obtained from cell-free supernatant of B. subtilis K1 revealed the presence of compounds with molecular mass ions in the range, m/z 1,028–1,109, m/z 994–1,065 and m/z 1,421–1,566, which were assigned as iturins, surfactins and fengycins, respectively (Kowall et al 1998; Vater et al 2002; Williams and Brodbelt 2004; Gong et al 2006; Romero et al 2007; Pathak et al 2012)
Summary
Endophytes are microorganisms which reside within plant tissues without causing any harm to host and they represent potential source of novel bioactive metabolites (Strobel 2006). The non-ribosomally synthesized lipopeptide antibiotics are well documented for their antifungal, antibacterial, antiviral, insecticidal, antitumor and surfactant activities (Winkelmann et al 1983; Vanittnakom et al 1986; Vollenbroich et al 1997; Kracht et al 1999; Stein 2005; Ongena and Jacques 2007; Kim et al 2007, 2010; Geetha et al 2010). The fungal antagonistic activity of Bacillus sp. has been correlated with the amount as well heterogeneity in the production of lipopeptides (Nagorska et al 2007; Ongena and Jacques 2007; Pecci et al 2010)
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