Journal of Biotechnology 125 (2006) 513515

Short communication

Second stage production of iturin A by induced germination of Bacillus subtilis RB14

Mohammad Shahedur Rahman , Takashi Ano, Makoto Shoda
Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan Received 14 December 2005; received in revised form 22 February 2006; accepted 13 March 2006

Abstract Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was dened as second stage production. 2006 Elsevier B.V. All rights reserved.
Keywords: Bacillus subtilis; Iturin A; Induced germination; Second stage production

In recent years, there is a considerable interest in using Bacillus subtilis producing lipopeptide antibiotics like iturin A and surfactin as a biocontrol agent due to its antagonistic and repressive activity over plant pathogens (Phae and Shoda, 1990; Bais et al., 2004). Iturin A is a valuable pesticide drug for its large antifungal spectrum while it has low toxicity and low allergic effect (Delcambe et al., 1977). Iturin A, is a cyclic lipopeptide molecule produced nonribosomally by several strains of B. subtilis where R is the
Corresponding author. Tel.: +81 45 924 5268; fax: +81 45 924 5976. E-mail address: rahman@res.titech.ac.jp (M.S. Rahman).

aliphatic chain of the -amino acid. In our laboratory the wild type B. subtilis RB14 was rst characterized as a superior dual producer of iturin A and surfactin with suppressive effect against various plant diseases (Hiraoka et al., 1992). In the production of lipopeptides in the submerged fermentation, sporulation of B. subtilis is the natural phenomenon as it occurs following starvation. A series of changes in the transcription pattern ultimately results in the development of endospore (Errington, 1993; Piggot and Coote, 1976; Stragier and Losick, 1996). Previous studies revealed that the regulation of peptide antibiotics is carried out by the sporulation gene product Spo0A that regulates the biosynthesis of peptide antibiotics (Marahiel et al., 1993). The close relation-

0168-1656/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.03.016

514

M.S. Rahman et al. / Journal of Biotechnology 125 (2006) 513515

ship between the sporulation factors and lipopeptide antibiotic productions is attributed to the stage of the culture and production order as well. Though their productions are carried out at different growth phases during the culture incubation, the resting stage-sporulation nally terminates the production of these lipopeptides. The aim of this study is induction of re-germination to activate the spores metabolically for the re-production of iturin A in an old culture of B. subtilis RB14. Submerged fermentation of B. subtilis RB14 was carried out in 40 ml of lipopeptide antibiotic production medium in a 200 ml Erlenmeyer ask. This medium consists of 8% Polypepton S (Nippon Pharmaceuticals Co., Tokyo), 6.7% maltose, K2 HPO4 0.5%, MgSO4 7H2 O 0.05%, 25 mg/l FeSO4 7H2 O, 22 mg/l MnSO4 7H2 O and 184 mg/l CaCl2 . Maltose and minerals were purchased from Wako Pure Chemical Industries, Osaka. The asks were incubated at 30 C with a shaking speed of 120 strokes per min. As shown in Fig. 1, iturin A production became saturated on its fourth day of incubation when the nutrient was depleted and almost entire cell population became spores which was determined by heat sensitivity test by heating the spore aliquot at 80 C for 10 min and plated on LB agar (1% Polypepton (Nippon Pharmaceuticals Co., Tokyo), 0.5% yeast extract (Oriental Co., Tokyo), 0.5% NaCl, pH 7.0 supplemented with 2% agar) plate. To induce germination, the culture was supplemented with fresh Polypepton S (8% of culture volume, w/v). Powdered Polypepton S was weighed and

transferred into sterilized and dried fresh Erlenmeyer asks covered by silicon caps and used to supplement as it is to reduce the volume effect on productivity. The dry nutrient preparation was sterilized by using microwave oven (1000 W) for 1.5 min twice and transferred into the culture ask. Hundred percent sterilization by microwave oven was conrmed by colony appearance after spreading the aliquot of the medium on the LB-agar plate. Quantication of iturin A was modied from the previous method (Phae et al., 1990) that was performed by high performance liquid chromatography (HPLC) with mobile phase of acetonitrile and 10 mM ammonium acetate, (35:65, v/v) through ODS column (chromolith performance RP-18e 1004.6 mm, Merck KGaA, Darmstadt, Germany) operated at 30 C, at a ow rate of 2 ml/min. The productivity of iturin A was determined after another 4-day incubation following nutrient supplementation but no further production of iturin A was observed. Due to the dormancy stage of the spores or due to the effect of unknown repressor molecules spores did not become metabolically active in this condition. The culture was then supplemented with Polypepton S followed by a sublethal heat shock of 70 C for 10 min to activate the spores. By this activation, the production prole of iturin A changed and productivity increased as shown in Fig. 2. The simple interpretation that comes out for the increased productivity of iturin A is the generation of metabolically active cells in the old culture, after

Fig. 1. Iturin A productivity ( ) and cfu for total cellular frequency ( ) and sporulation () of B. subtilis RB14 in lipopeptide antibiotic production medium. Repetitions of these experiments gave the values within 20% of the values shown (n = 24).

Fig. 2. Time course of second stage production of iturin A. White arrow indicates the treatment moment for induced germination. Symbols ( ) and ( ) represent with and without treatment (heat and Polypepton S supplementation) runs, respectively. P indicates the improvement of production of iturin A by second stage production.

M.S. Rahman et al. / Journal of Biotechnology 125 (2006) 513515

515

heating and Polypepton S supplementation. Though the productivity of iturin A increased but productivity of another lipopeptide surfactin remained unchanged for this treatment (data not shown). It is well accepted that production of surfactin is comparatively earlier than the other lipopeptide antibiotics from the same organism (Tsuge et al., 1995; Stein, 2005). Therefore, it can be said that second stage production can be applicable for the lipopeptides produced in the later phase of growth of B. subtilis. Since the late 70s of last century several reports explained germination in the presence of nutrients. B. subtilis spores readily germinate in the presence of nutrients where a variety of small molecules can also trigger spore germination (Gould, 1969; Setlow, 1983; Wuytack et al., 2000; Paidhungat and Setlow, 2002). In all of the previously reported studies, germination was analyzed in freshly prepared medium and no experiments for the spores in an old culture medium have been reported. In this study, we have conrmed that only nutrient addition cannot improve the production of iturin A in the old culture medium and it can overcome successfully by heat activation. Heat-activated spores in the old culture medium were germinated and led to another round of production of the lipopeptide antibiotic iturin A. This work demonstrated that metabolically active cells derived from induced germination of the RB14 spores were responsible for re-production of iturin A after their rst metabolic stage when iturin A was produced and the cells became spore. The second time production of the secondary metabolite iturin A by the same cells is dened as second stage production, which is a new tool that metabolically activates the spores in the old production medium. Considering its production improvement characteristic, second stage production has signicant potentiality for application for production improvement of natural or synthetic metabolites; those are produced at their late transition or stationary phase of B. subtilis or other spore forming bacteria.

References
Bais, H.P., Fall, R., Vivanco, M., 2004. Biocontrol of Bacillus subtilis against infection of arabidopsis roots by Pseudomonas syringae is facilitated by biolm formation and surfactin production. Plant Physiol. 134, 307319. Delcambe, L., Peypoux, F., Besson, F., Guinand, M., Michel, G., 1977. Structure of iturin and iturin-like substances. In: Biochemical Society Transactions, 569th Meeting, Sussex, UK. Errington, J., 1993. Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol. Rev. 57, 133. Gould, G.W., 1969. Germination. In: Gould, G.W., Hurst, A. (Eds.), The Bacterial Spore. Academic press, London, England, pp. 359396. Hiraoka, H., Asaka, O., Ano, T., Shoda, M., 1992. Characterization of Bacillus subtilis RB14, coproducer of peptide antibiotics iturin A and surfactin. J. Gen. Appl. Microbiol. 38, 635640. Marahiel, M.A., Nakano, M.M., Zuber, P., 1993. Regulation of peptide antibiotic production in Bacillus. Mol. Microbiol. 7, 631636. Paidhungat, M., Setlow, P., 2002. Spore germination and outgrowth. In: Sonenshein, A.L., Hoch, J.A., Losick, R. (Eds.), Bacillus subtilis and its Closest Relatives. ASM Press, Washington, DC, pp. 537548. Phae, C.G., Shoda, M., Kuboda, H., 1990. Suppressive effect of Bacillus subtilis and its products on phytopathogenic microorganisms. J. Ferment. Bioeng. 69, 17. Phae, C.G., Shoda, M., 1990. Expression of suppressive effect of Bacillus subtilis on phytopathogens in inoculated composts. J. Ferment. Bioeng. 70, 409414. Piggot, P.J., Coote, J.G., 1976. Genetic aspects of bacterial endospore formation. Bacteriol. Rev. 40, 908962. Setlow, P., 1983. Germination and outgrowth. In: Gould, G.W., Hurst, A. (Eds.), The Bacterial Spore, vol. II. Academic press, London, England, pp. 211254. Stein, T., 2005. Bacillus subtilis antibiotics: structures, syntheses and specic functions. Mol. Microbiol. 56, 845857. Stragier, P., Losick, R., 1996. Molecular genetics of sporulation in Bacillus subtilis. Annu. Rev. Genet. 30, 297341. Tsuge, K., Ano, T., Shoda, M., 1995. Characterization of Bacillus subtilis YB8 coproducer of lipopeptides surfactin and plipastatin B1. J. Gen. Appl. Microbiol. 41, 541545. Wuytack, E.Y., Soons, J., Poschet, F., Michiels, C.W., 2000. Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores. Appl. Environ. Microbiol. 66, 257 261.