A bacterium, which showed antifungal activity against G. gloeosporioides, was isolated from rice straw, and was identified as B. subtilis from its morphological and physiological characteristics. From the 16S rDNA sequence analyses, the isolate showed 98% similarity to B. subtilis ATCC 21331 (Fig. 1). The 16S rDNA sequence of the isolate has been deposited in the GenBank database, under the accession number AY207468.
In order to study the antifungal compounds production during the growth of the B. subtilis KS03, the antibiotic activity present in the cell-free samples, taken at various time intervals, was measured. The antifungal compounds produced by the B. subtilis strain KS03 near the end of the stationary phase were detected by bioassays after 32 h of culturing. At the death phase (60 h), the production of the antifungal compounds was at its highest with no significant change in the levels before/after that time (Fig. 2).
From extraction experiments with various solvents, antifungal activity was detected in butanol, suggesting it to be highly hydrophobic. Extraction of the supernatants of the B. subtilis KS03 cultures with butanol resulted in the transfer of the antifungal activity into the organic phase. Further purification was achieved by DEAE Sepharose chromatography and preparative TLC. The Fourier transform infrared (FTIR) spectra of the purified compound had broad bands, centering around 3300 cm−1, indicative of an associated NH, and amide I and II bands at 1647 and 1510 cm−1, respectively (Fig. 3). A MALDI mass spectral analysis revealed a cluster containing three molecules that was observed at m/z 1043, 1057 and 1071 (data not shown). These peaks differ by 14 Da, suggesting a series of homologous molecules having different length of fatty acid chain, i.e. CH2=14 Da. The MALDI MS/MS spectrum of the major protonated molecule [M+H]+, observed at m/z 1043, was exactly the same as that of the [M+H]+ of authentic, commercially available iturin A (Fig. 4). The three molecules were antifungal lipopeptides with the structure Pro-Asn-Ser-βAA-Asn-Tyr-Asn-Gln (βAA representing the β-amino acid). The lower-mass region of the MS/MS spectra indicated peaks corresponding to the immonium ions (H2N+=CH-R) of the individual constituent amino acids, namely Ser (m/z 60), Pro (m/z 70), Gln (m/z 84), Asn (m/z 87) and Tyr (m/z 136). The β-amino acid also had an immonium ion (H2N+=CH-C11H23) at m/z 184. The main linear acylium ions of iturin should be Pro-Asn-Ser-βAA-Asn-Tyr-Asn-Gln-CO+, due to the presence of proline in the molecule. The collision-induced dissociation (CID) spectra showed fragment ions indicative of the amino acid sequence of iturin. Especially, as shown in Fig. 4, there were two series of b-type and complementary y-type ions contained, due to the cleavages of the amide bonds between the two neighboring amino acid residues along the peptide backbone. There were isomers of each iturin, due to the different structures of the fatty acid side chain, such as the n-, iso-, or anteiso-forms. Iturin A has the sequence βAA-Asn-Tyr-Asn-Gln-Pro-Asn-Ser. In nature, iturin A is produced as a mixture of up to eight isomers, named iturin A1 to A8. The major antifungal compound purified from B. subtilis KS03 has the same sequence and molecular mass as iturin A2.
Iturins have been used for the biological control of a variety of plant pathogens, such as Monilinia fructicola, Ophiostoma ulmi, Aspergillus flavus, and Rhizoctonia solani. In this study, we have described the production and purification of iturin A by the biological control agent B. subtilis strain KS03 which is active against the anthracnose disease fungus G. gloeosporioides.