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Effect of temperature on production of lipopeptide antibiotics, iturin A and surfactin by a dual producer, Bacillus subtilis RB14, in solid-state fermentation
JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 80, No. 5, 517-519. 1995
Effect of Temperature on Production of Lipopeptide Antibiotics, Iturin A and Surfactin by a Dual Producer, Bacillus subtilis RB14, in Solid-State Fermentation AKIHIRO Research Laboratory
OHNO,
TAKASHI
ANO, AND MAKOTO
SHODA*
of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan Received
22 May 1995/Accepted
5 August
1995
of the production of iturin A and surfactin by a dual producer, Bacillus subtilis of okara was investigated. The optimal temperature for iturin A was 25’C, while that for surfactin was 37’C, in spite of their dependency on a common gene, @a-14. When the effect of temperature on the relative ratios of the amount of the five homologues of iturin A to the total iturin A produced by RB14 was investigated, only the ratios of homologues having p-amino acid residues with normal aliphatic chains were affected and the ratio of the homologue with longer normal (C16-) chain increased as the temperature was increased. Temperature
dependency
RB14, in the solid-state fermentation
[Key words:
iturin A, surfactin, Bacillus subtilis, solid state fermentation] only genes related to surfactin production have been studied by other researchers (14-18). Although coproduction of these two antibiotics was first reported by Sandrin et al. (8), optimal conditions for production of surfactin (6, 19-21) and iturin A (11) have been investigated separately only in submerged fermentation. We were first to show that solid-state fermentation (SSF) is effective for the production of iturin A (22). There have been no reports on the production of the two compounds in the SSF by a dual producer. In this work, we report the effect of temperature on the production of iturin A and surfactin by this dual producer, B. subtilis RB14, in the SSF of soybean curd residue, “okara,” a by-product of soybean curd known as tofu, which is used as a solid substrate. Solid-state fermentation was conducted as follows: okara with a moisture content of 77% was provided by a commercial tofu supplier (Marusho Shoten, Tokyo). Fifteen grams of okara were placed in a 100 ml Erlenmeyer flask, the mouth of which was stoppered with a cotton plug, and autoclaved twice at 120°C for 30 min at an interval of 8-12 h to kill the spore-forming microorganisms inhabiting the material. After autoclaving, 833 ~1 of 0.45 g/ml glucose, 75 ~1 of 1 M KH2P04, 225 /*l of 1 M MgS04 and 367 ~1 of deionized distilled water were aseptically added for the fortification of nutrients (20), and 3 ml of a preculture of RB14 grown in the no. 3S medium [log Polypepton S (Nippon Pharmaceuticals Co., Tokyo), 10 g glucose, 1 g KH2P04, and 0.5 g MgS04 in 1 I distilled water] was inoculated. The flasks were immersed in a water incubator for 48 h without shaking. Cultivation temperature was varied from 23, 25, 30, 37, 42, to 48°C in accordance with the purpose. Iturin A and surfactin were extracted from the cultivated okara with methanol. The extract was then centrifuged, and the supernatant was filtered prior to the injection into high performance liquid chromatography (HPLC) (either 880 PU or TRI ROTAR-V, Japan Spectroscopic Co., Tokyo) using a UV detector set at 205 nm (870 UV or 875 UV, Japan Spectroscopic Co., Tokyo) to determine the concentrations of iturin A and
Due to its potential for use as a biological pesticide (1, 2), we isolated Bacillus subtilis, from the environment (3, 4). A new isolate of B. subtiLts from compost, named RB14, showed stronger inhibitory activity against various phytopathogens in vitro (5) than B. subtilis NB22 (1, 3, 4). We found that the strain RB14 produces cyclic lipopeptide antibiotics, iturin A and surfactin simultaneously. Although surfactin alone did not inhibit growth of phytopathogenic fungi, it significantly enhanced the antifungal activity of iturin A and this synergistic antifungal activity exhibited by the two compounds was first reported by us (5). This is probably due to the cytolytic activity of surfactin (6-8) which weakens cell membrane, enabling the easy attack of iturin A (1, 3, 9-l 1). The general structures of both compounds are as follows: Iturin A: RCHCH2CO+L-Asn+D-Tyr’D_Asn I NH+L-Ser+D-Asn+L-Pro+L-Glu Surfactin: R’CHCH2CO+L-Glu+L-Leu-tD-Leu b+L-Leu+D-Leu+L-Asp+D-VLl where R and R’ are aliphatic side chains. To elucidate the synthetic pathways of these two lipopeptides, we have developed a transformation system applicable to this wild strain of B. subtilis (12). We found a gene within RB14, named lpa-14, responsible for the production of surfactin in a derivative of Marburg 168 (13). When this gene was destroyed in the original strain, RB14, the productivities of the two lipopeptides were lost simultaneously. However, introduction of the intact Ipagene into the defective strain lead to the recovery of the dual productivity. Although the precise mechanisms of the production of each compounds by RB14 are unknown, lpa-14 is known to be involved in the biosyntheses of surfactin and iturin A (5). So far, * Corresponding
author. 517
OHNO
J. FERMENT. BIOENG.,
ET AL.
25
30
42
37
48
Temperature (“c)
23
25
30
37
Temperature (“C)
35
40
45
50
Temperature (“c)
FIG. 2. The ratios of the amount of iturin A homologue to the total amount of iturin A produced by B. subtilis RB14 in the solidstate fermentation of okaru. Numbers l-5 in the figure represent homologues of iturin A with n-Cr.,-, anteiso-C,,-, iso-C,,-, n-C16-, and iso-Ct6-&amino acid residues, respectively.
f
20
30
Time (II) FIG. 1. Effect of temperature on the production of iturin A and surfactin and on the growth of B. subtilis RB14 in solid-state fermentation of okara. (a) Concentrations of iturin A and surfactin after 48 h at different temperatures. Symbols: 3, iturin A; q , surfactin. (b) Ratio of the amount of iturin A and surfactin to their maximum amount produced at different temperatures. Symbols: A, iturin A; 0, surfactin. (c) Count of viable cells; 0, 25°C; 0, 30°C; n, 37’C; A, 42°C; and 0, 48°C.
surfactin. Calibration curves were prepared by using individually purified five peaks of iturin A (3, 4) and the commercial product of surfactin (Wako Pure Chemicals Industry, Osaka). Conditions for quantification by HPLC are as follows. For iturin A: mobile phase, acetonitrile/lO mM ammonium acetate=4/3 (v/v); column, 4.6 mm+ x 250 mm ODS-2; column temperature, 30°C; flow rate, l.Oml/min; and for surfactin: mobile phase, acetonitrile/3.8 mM trifluoroacetic acid = 4/l (v/v), the same column at the same temperature with that of iturin A was used; and flow rate, 1.5 ml/min. Figure 1 shows the effect of temperature on the production of iturin A and surfactin by RB14 and on the growth of RB14. Figure la shows the actual amounts of iturin A and surfactin produced. Figure lb shows relative productivity when the maximum value in Fig. 1 is taken to be 100%. Production of iturin A decreased as the temperature was increased. Surfactin production, in contrast, increased up to 37°C and then decreased beyond that temperature. Between 25°C and 37°C the production profiles of the two compounds were very different. Figure lc indicates that cell population varied within one order of magnitude among the temperatures
tested, and the final viable cell counts of RB14 at each temperature were proportional to the concentration of surfactin. This may correspond to the fact that surfactin is produced by RB14 in a growth-associated pattern. Although the precise metabolic pathways for the syntheses of iturin A and surfactin are not elucidated, their quite different temperature dependencies were clearly shown in our results (Figs. la and b). Although the same gene, lpa-14, coregulates the production of iturin A and surfactin (13, 23), our results suggest that the common pathways involving Lpa-14 protein are not significantly affected by the temperature, whereas the pathways that are specific for each compound are affected by the temperature. From the data showing the effect of temperature on the production of iturin A and surfactin, it is possible to produce iturin A and surfactin at different ratios in the fermented okara. As the optimum ratio of iturin A to surfactin observed to inhibit the growth of a phytopathogenie fungus, Fusarium oxysporum f. sp. lycopersici race Jl SUF119, is 1 to 4 when the concentration of surfactin is fixed at lOOmg/l (5), and the antiphytopathogenic activity may vary with the ratio of the concentrations of the two substances depending on plant pathogens; different activities as a biological pesticide against other phytopathogens can be expected from the solid culture fermented at different temperatures. Inhibitory activity of fermented okara as a whole against phytopathogens in soil with actual plant to examine its function as a biological pesticide to prevent plant diseases is now under investigation, There are five homologues of iturin A produced which can be distinguished and quantified using HPLC system described previously (2). In the course of investigation of temperature effects, we also found changes among the relative ratios of the five major homologues of iturin A as shown in Fig. 2. The effect of temperature on the ratio was marked for 1 and 4 that possess normal aliphatic chains. The ratio of peak 1 decreased as the temperature was increased whereas a completely opposite tendency was observed for peak 4. The effect of temperature was not so marked on 2, 3, and 5 that possess the
VOL. 80, 1995
NOTES
branched aliphatic chains such as iso or anteiso chains and their peak ratios of these peaks were almost constant at different temperatures. No information is available concerning the biosynthesis of iturin A, but the fatty acids of side chains can be postulated to be derived from the synthetic pathways of fatty acids of cellular membranes. The composition of cellular membrane fatty acids is altered by varying the growth temperature to maintain the proper membrane rigidity at a given temperature. It has been reported that the relative amount of long-chain fatty acids increases with increasing temperature for five Bacillus strains (24) as well as Clostridia (23, mainly because the longer the chain of a fatty acid is, the higher the melting temperature of the fatty acid becomes. In this experiment, the decrease in relative ratio of 1 with increasing temperature is similar to the result mentioned above. However, it is interesting that the ratio of 1 consisting of n-Cl.,-/-amino acid residue decreased with increasing temperature while that of 4 consisting of n-C&/?-amino acid residue increased. This may indicate that there is some specific metabolic change that occurs in the RB14 intracellularly. Although the total amount of iturin A produced decreased as temperature was increased, the efficacy of the activity of the fermented okara as a biological pesticide to inhibit plant pathogens, and thus to prevent plant diseases, may not vary so significantly mainly because the longer the side-chains of an iturin A homologue is, the lower the minimum inhibitory concentration for various plant pathogens becomes, as has been reported previously (3). Not only did we reveal the effect of temperature on iturin A and surfactin production, we also showed the effect of temperature on the relative ratios among the five iturin A homologues. Precise mechanisms of the temperature dependencies described above should be explained by biochemical and/or genetic studies.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
REFERENCES 1. Phae, C. G., Shoda, M., Kita, N., Nakano, M., and Ushiyama, K.: Biological control of crown and root rot and bacterial wilt of tomato by Bacillus subtilis NB22. Ann. Phytopath. Sot. Japan, 58, 329-339 (1992). 2. Boer, A. S. and Diderichsen, B.: On the safety of Bacillus subtilis and Bacillus amyloliquefaciens: a review. Appl. Microbial. Biotechnol., 36, l-4 (1991). 3. Phae, C. G., Shoda, M., and Kubota, H.: Suppressive effect of Bacillus subtilis and its products on phytopathogenic microorganisms. J. Ferment. Bioeng., 69, l-7 (1990). 4. Phae, C. G., Sasaki, M., Shoda, M., and Kubota, H.: Characteristics of Bacillus subtilis suppressive to phytopathogens isolated from composts. Soil. Sci. Plant Nutr., 36, 575-586 (1990). 5. Hiraoka, H., Asaka, O., Ano, T., and Shoda, M.: Characterization of Bacillus subtilis RB14, coproducer of peptide antibiotics iturin A and surfactin. J. Gen. Appl. Microbial., 38, 635640 (1992). a crys6. Arima, K., Kakinuma, A., and Tamura, G.: Surfactin, talline peptidelipid surfactant produced by BaciNus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun., 31, 488-494 (1968). 7. Magetdana, R., Thimon, L., Peypoux, F., and Ptak, M.: Surfactin/iturin A interactions may explain the synergistic effect of
19.
20.
21.
22.
23.
24.
25.
519
surfactin on the biological properties of iturin A. Biochimie, 74, 1047-1051 (1992). Sandrin, C., Peypoux, F., and Michel, G.: Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis. Biotechnol. Appl. Biochem., 12, 370-375 (1990). Bernheimer, A. W. and Avigad, L. S.: Nature and properties of a cytolytic agent produced by Bacillus subtilis. J. Gen. Microbial., 61, 361-369 (1970). Klich, M. A., Lax, A. R., and Bland, J. M.: Inhibition of some mycotoxigenic fungi by iturin A, a peptidolipid produced by Bacillus subtilis. Mycopathologia, 116, 77-80 (1991). Phae, C. G. and Shoda, M.: Investigation of optimal conditions for foam separation of iturin, an antifungal peptide produced by Bacillus subtilis. J. Ferment. Bioeng., 71, 118-121 (1991). Matsuno, Y., Hitomi, T., Ano, T., and Sboda, M.: Transformation of Bacillus subtilis, antifungal-antibiotic iturin producers with isolated antibiotic resistance plasmids. J. Gen. Appl. Microbial., 38, 13-21 (1992). Hiraoka, H., Ano, T., and Sboda, M.: Molecular cloning of a gene responsible for the biosynthesis of the lipopeptide antibiotics iturin and surfactin. J. Ferment. Bioeng., 74, 323-326 (1992). Cosmina, P., Rodriguez, F., Deferra, F., Grandi, G., Perego, M., Venema, G., and Vansinderen, D.: Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Mol. Microbial., 8, 821-831 (1993). Morlkawa, M., Ito, M., and Imanaka, T.: Isolation of a new surfactin producer Bacillus pumilus A-l, and cloning and nucleotide sequence of the regulator gene, PSF-1. J. Ferment. Bioeng., 74, 255-261 (1992). Nakano, M. M., Marahiel, M. A., and Zuber, P.: Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtihs. J. Bacterial., 170, 5662-5668 (1988). Nakano, M. M., Magnuson, R., Myers, A., Curry, J., Grossman, A. D., and Zuber, P.: srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis. J. Bacterial., 173, 1770-1778 (1991). Nakano, M. M., Corbell, N., Besson, J., and Zuber, P.: Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtifis. Mol. Gen. Genet., 232, 313-321 (1992). Cooper, D. G., MacDonald, C. R., Duff, S. J. B., and Kosaric, N.: Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Appl. Environ. Microbial., 42, 408-412 (1981). Obno, A., Ano, T., and Shoda, M.: Production of a lipopeptide antibiotic surfactin with recombinant Bacillus subtilis. Biotechnol. Lett., 14, 1165-1168 (1992). Sheppard, J. D. and Mulligan, N.: The production of surfactin by Bacillus subtilis grown on peat hydrolysate. Appl. Microbiol. Biotechnol., 27, 110-I 16 (1987). Ohno, A., Aao, T., and Shoda, M.: Production of antifungal peptide antibiotic iturin by Bacillus subtilis NB22 in solid state fermentation. J. Ferment. Bioeng., 75, 23-27 (1993). Huang, C.-C., Ano, T., and Shoda, M.: Nucleotide sequence and characteristics of the gene, lpa-14, responsible for biosynthesis of the lipopeptide antibiotics iturin A and surfactin from B. subti/is RB14. J. Ferment. Bioeng., 76, 445-450 (1993). Kaneda, T.: Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbial. Rev., 55, 288-302 (1991). Ghan, M., Himes, R. H., and Akagi, J. M.: Fatty acid composition of thermophilic, mesophilic, and psychrophilic Clostridia. J. Bacterial., 106, 876881 (1971).
Effect of Temperature on Production of Lipopeptide Antibiotics, Iturin A and Surfactin by a Dual Producer, Bacillus subtilis RB14, in Solid-State Fermentation AKIHIRO Research Laboratory
OHNO,
TAKASHI
ANO, AND MAKOTO
SHODA*
of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan Received
22 May 1995/Accepted
5 August
1995
of the production of iturin A and surfactin by a dual producer, Bacillus subtilis of okara was investigated. The optimal temperature for iturin A was 25’C, while that for surfactin was 37’C, in spite of their dependency on a common gene, @a-14. When the effect of temperature on the relative ratios of the amount of the five homologues of iturin A to the total iturin A produced by RB14 was investigated, only the ratios of homologues having p-amino acid residues with normal aliphatic chains were affected and the ratio of the homologue with longer normal (C16-) chain increased as the temperature was increased. Temperature
dependency
RB14, in the solid-state fermentation
[Key words:
iturin A, surfactin, Bacillus subtilis, solid state fermentation] only genes related to surfactin production have been studied by other researchers (14-18). Although coproduction of these two antibiotics was first reported by Sandrin et al. (8), optimal conditions for production of surfactin (6, 19-21) and iturin A (11) have been investigated separately only in submerged fermentation. We were first to show that solid-state fermentation (SSF) is effective for the production of iturin A (22). There have been no reports on the production of the two compounds in the SSF by a dual producer. In this work, we report the effect of temperature on the production of iturin A and surfactin by this dual producer, B. subtilis RB14, in the SSF of soybean curd residue, “okara,” a by-product of soybean curd known as tofu, which is used as a solid substrate. Solid-state fermentation was conducted as follows: okara with a moisture content of 77% was provided by a commercial tofu supplier (Marusho Shoten, Tokyo). Fifteen grams of okara were placed in a 100 ml Erlenmeyer flask, the mouth of which was stoppered with a cotton plug, and autoclaved twice at 120°C for 30 min at an interval of 8-12 h to kill the spore-forming microorganisms inhabiting the material. After autoclaving, 833 ~1 of 0.45 g/ml glucose, 75 ~1 of 1 M KH2P04, 225 /*l of 1 M MgS04 and 367 ~1 of deionized distilled water were aseptically added for the fortification of nutrients (20), and 3 ml of a preculture of RB14 grown in the no. 3S medium [log Polypepton S (Nippon Pharmaceuticals Co., Tokyo), 10 g glucose, 1 g KH2P04, and 0.5 g MgS04 in 1 I distilled water] was inoculated. The flasks were immersed in a water incubator for 48 h without shaking. Cultivation temperature was varied from 23, 25, 30, 37, 42, to 48°C in accordance with the purpose. Iturin A and surfactin were extracted from the cultivated okara with methanol. The extract was then centrifuged, and the supernatant was filtered prior to the injection into high performance liquid chromatography (HPLC) (either 880 PU or TRI ROTAR-V, Japan Spectroscopic Co., Tokyo) using a UV detector set at 205 nm (870 UV or 875 UV, Japan Spectroscopic Co., Tokyo) to determine the concentrations of iturin A and
Due to its potential for use as a biological pesticide (1, 2), we isolated Bacillus subtilis, from the environment (3, 4). A new isolate of B. subtiLts from compost, named RB14, showed stronger inhibitory activity against various phytopathogens in vitro (5) than B. subtilis NB22 (1, 3, 4). We found that the strain RB14 produces cyclic lipopeptide antibiotics, iturin A and surfactin simultaneously. Although surfactin alone did not inhibit growth of phytopathogenic fungi, it significantly enhanced the antifungal activity of iturin A and this synergistic antifungal activity exhibited by the two compounds was first reported by us (5). This is probably due to the cytolytic activity of surfactin (6-8) which weakens cell membrane, enabling the easy attack of iturin A (1, 3, 9-l 1). The general structures of both compounds are as follows: Iturin A: RCHCH2CO+L-Asn+D-Tyr’D_Asn I NH+L-Ser+D-Asn+L-Pro+L-Glu Surfactin: R’CHCH2CO+L-Glu+L-Leu-tD-Leu b+L-Leu+D-Leu+L-Asp+D-VLl where R and R’ are aliphatic side chains. To elucidate the synthetic pathways of these two lipopeptides, we have developed a transformation system applicable to this wild strain of B. subtilis (12). We found a gene within RB14, named lpa-14, responsible for the production of surfactin in a derivative of Marburg 168 (13). When this gene was destroyed in the original strain, RB14, the productivities of the two lipopeptides were lost simultaneously. However, introduction of the intact Ipagene into the defective strain lead to the recovery of the dual productivity. Although the precise mechanisms of the production of each compounds by RB14 are unknown, lpa-14 is known to be involved in the biosyntheses of surfactin and iturin A (5). So far, * Corresponding
author. 517
OHNO
J. FERMENT. BIOENG.,
ET AL.
25
30
42
37
48
Temperature (“c)
23
25
30
37
Temperature (“C)
35
40
45
50
Temperature (“c)
FIG. 2. The ratios of the amount of iturin A homologue to the total amount of iturin A produced by B. subtilis RB14 in the solidstate fermentation of okaru. Numbers l-5 in the figure represent homologues of iturin A with n-Cr.,-, anteiso-C,,-, iso-C,,-, n-C16-, and iso-Ct6-&amino acid residues, respectively.
f
20
30
Time (II) FIG. 1. Effect of temperature on the production of iturin A and surfactin and on the growth of B. subtilis RB14 in solid-state fermentation of okara. (a) Concentrations of iturin A and surfactin after 48 h at different temperatures. Symbols: 3, iturin A; q , surfactin. (b) Ratio of the amount of iturin A and surfactin to their maximum amount produced at different temperatures. Symbols: A, iturin A; 0, surfactin. (c) Count of viable cells; 0, 25°C; 0, 30°C; n, 37’C; A, 42°C; and 0, 48°C.
surfactin. Calibration curves were prepared by using individually purified five peaks of iturin A (3, 4) and the commercial product of surfactin (Wako Pure Chemicals Industry, Osaka). Conditions for quantification by HPLC are as follows. For iturin A: mobile phase, acetonitrile/lO mM ammonium acetate=4/3 (v/v); column, 4.6 mm+ x 250 mm ODS-2; column temperature, 30°C; flow rate, l.Oml/min; and for surfactin: mobile phase, acetonitrile/3.8 mM trifluoroacetic acid = 4/l (v/v), the same column at the same temperature with that of iturin A was used; and flow rate, 1.5 ml/min. Figure 1 shows the effect of temperature on the production of iturin A and surfactin by RB14 and on the growth of RB14. Figure la shows the actual amounts of iturin A and surfactin produced. Figure lb shows relative productivity when the maximum value in Fig. 1 is taken to be 100%. Production of iturin A decreased as the temperature was increased. Surfactin production, in contrast, increased up to 37°C and then decreased beyond that temperature. Between 25°C and 37°C the production profiles of the two compounds were very different. Figure lc indicates that cell population varied within one order of magnitude among the temperatures
tested, and the final viable cell counts of RB14 at each temperature were proportional to the concentration of surfactin. This may correspond to the fact that surfactin is produced by RB14 in a growth-associated pattern. Although the precise metabolic pathways for the syntheses of iturin A and surfactin are not elucidated, their quite different temperature dependencies were clearly shown in our results (Figs. la and b). Although the same gene, lpa-14, coregulates the production of iturin A and surfactin (13, 23), our results suggest that the common pathways involving Lpa-14 protein are not significantly affected by the temperature, whereas the pathways that are specific for each compound are affected by the temperature. From the data showing the effect of temperature on the production of iturin A and surfactin, it is possible to produce iturin A and surfactin at different ratios in the fermented okara. As the optimum ratio of iturin A to surfactin observed to inhibit the growth of a phytopathogenie fungus, Fusarium oxysporum f. sp. lycopersici race Jl SUF119, is 1 to 4 when the concentration of surfactin is fixed at lOOmg/l (5), and the antiphytopathogenic activity may vary with the ratio of the concentrations of the two substances depending on plant pathogens; different activities as a biological pesticide against other phytopathogens can be expected from the solid culture fermented at different temperatures. Inhibitory activity of fermented okara as a whole against phytopathogens in soil with actual plant to examine its function as a biological pesticide to prevent plant diseases is now under investigation, There are five homologues of iturin A produced which can be distinguished and quantified using HPLC system described previously (2). In the course of investigation of temperature effects, we also found changes among the relative ratios of the five major homologues of iturin A as shown in Fig. 2. The effect of temperature on the ratio was marked for 1 and 4 that possess normal aliphatic chains. The ratio of peak 1 decreased as the temperature was increased whereas a completely opposite tendency was observed for peak 4. The effect of temperature was not so marked on 2, 3, and 5 that possess the
VOL. 80, 1995
NOTES
branched aliphatic chains such as iso or anteiso chains and their peak ratios of these peaks were almost constant at different temperatures. No information is available concerning the biosynthesis of iturin A, but the fatty acids of side chains can be postulated to be derived from the synthetic pathways of fatty acids of cellular membranes. The composition of cellular membrane fatty acids is altered by varying the growth temperature to maintain the proper membrane rigidity at a given temperature. It has been reported that the relative amount of long-chain fatty acids increases with increasing temperature for five Bacillus strains (24) as well as Clostridia (23, mainly because the longer the chain of a fatty acid is, the higher the melting temperature of the fatty acid becomes. In this experiment, the decrease in relative ratio of 1 with increasing temperature is similar to the result mentioned above. However, it is interesting that the ratio of 1 consisting of n-Cl.,-/-amino acid residue decreased with increasing temperature while that of 4 consisting of n-C&/?-amino acid residue increased. This may indicate that there is some specific metabolic change that occurs in the RB14 intracellularly. Although the total amount of iturin A produced decreased as temperature was increased, the efficacy of the activity of the fermented okara as a biological pesticide to inhibit plant pathogens, and thus to prevent plant diseases, may not vary so significantly mainly because the longer the side-chains of an iturin A homologue is, the lower the minimum inhibitory concentration for various plant pathogens becomes, as has been reported previously (3). Not only did we reveal the effect of temperature on iturin A and surfactin production, we also showed the effect of temperature on the relative ratios among the five iturin A homologues. Precise mechanisms of the temperature dependencies described above should be explained by biochemical and/or genetic studies.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
REFERENCES 1. Phae, C. G., Shoda, M., Kita, N., Nakano, M., and Ushiyama, K.: Biological control of crown and root rot and bacterial wilt of tomato by Bacillus subtilis NB22. Ann. Phytopath. Sot. Japan, 58, 329-339 (1992). 2. Boer, A. S. and Diderichsen, B.: On the safety of Bacillus subtilis and Bacillus amyloliquefaciens: a review. Appl. Microbial. Biotechnol., 36, l-4 (1991). 3. Phae, C. G., Shoda, M., and Kubota, H.: Suppressive effect of Bacillus subtilis and its products on phytopathogenic microorganisms. J. Ferment. Bioeng., 69, l-7 (1990). 4. Phae, C. G., Sasaki, M., Shoda, M., and Kubota, H.: Characteristics of Bacillus subtilis suppressive to phytopathogens isolated from composts. Soil. Sci. Plant Nutr., 36, 575-586 (1990). 5. Hiraoka, H., Asaka, O., Ano, T., and Shoda, M.: Characterization of Bacillus subtilis RB14, coproducer of peptide antibiotics iturin A and surfactin. J. Gen. Appl. Microbial., 38, 635640 (1992). a crys6. Arima, K., Kakinuma, A., and Tamura, G.: Surfactin, talline peptidelipid surfactant produced by BaciNus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun., 31, 488-494 (1968). 7. Magetdana, R., Thimon, L., Peypoux, F., and Ptak, M.: Surfactin/iturin A interactions may explain the synergistic effect of
19.
20.
21.
22.
23.
24.
25.
519
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