Plasmid transformation in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer

A transformation system with plasmids was developed for Bacillus subtilis NB22, an antibiotic iturin producing strain. Treatment of B. subtilis NB22 with 4 M KCl was effective for the induction of competence, followed by uptake of plasmid DNA in the presence of polyethylene glycol. The efficiency of transformation of this bacterium with pC194 and pUB110 was 4.1 X 10(3) and 1.5 X 10(3) transformants per micrograms DNA, respectively and the transformation frequency was 3.3 X 10(-3) and 7.2 X 10(-4), transformants per viable cell, respectively. This method was much faster and three orders of magnitude more efficient in transformation efficiency than protoplast transformation methods.     

Characteristics of plasmid stability in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer

The stability of plasmids pC194 and pUB110 was investigated in an antifungal antibiotic iturin producer, Bacillus subtilis NB22. Although plasmid pC194 was maintained stably over a hundred generations in five successive cultivations in iturin-production no. 3 medium, significant curing was observed when the cultivation was prolonged for 10 d in the same medium. When the transformant of pC194 was cultivated in Schaeffer's sporulation medium, drastic curing took place in accordance with the occurrence of sporulation, even in the presence of an antibiotic for selective pressure. In the case of pUB110, in sharp contrast to the result with pC194, high stability was observed in the sporulation medium, but significant curing was observed during prolonged incubation in no. 3 medium.     

High-efficiency transformation of Bacillus subtilis NB22, an antifungal antibiotic iturin producer,by electroporation

High-voltage electroporation was used to transform Bacillus subtilis NB22, an antifungal antibiotic producer, reaching the efficiency of 10⁷ transformants/μg plasmid DNA. Transformation frequency was dependent on the composition of the electroporation solution, the electrical field strength and the cell concentration. Addition of polyethylene glycol (PEG) and mannitol in the transformation solution was critical for a high efficiency of transformation.     

Production of the antifungal peptide antibiotic,iturin by Bacillus subtilis NB22 in solid state fermentation

Production of iturin, an antifungal peptide effective at suppressing phytopathogens, by Bacillus subtilis NB22 was investigated in solid state fermentation (SSF) using soy bean curd residue (okara). In scale-up from 15 g to 3 kg, aeration, temperature, and moisture content were controlling factors for the efficient production of iturin. It was found that solid state fermentation was 6–8 times more efficient with respect to iturin productivity than submerged fermentation on the basis of unit wet weight. Higher productivity in selective production of specific components of iturin which are stronger inhibitors of plant pathogens was also confirmed in SSF.     

Production of antifungal antibiotic,iturin in a solid state fermentation byBacillus subtilis NB22 using wheat bran as a substrate

Summary Production of a family of lipopeptide antibiotic, iturin byB. subtilis NB22, in solid state fermentation (SSF) of wheat bran (WB) was investigated. The amount of iturin produced per unit weight of wet substrate was 5–6 times more than that in the submerged fermentation (SMF). SSF enabled to produce a homologue of iturin with strong antibiotic activity in a larger fraction compared with the SMF.     

Characterization of iturin synthetase in the wild-type Bacillus subtilis strain producing iturin and in an iturin deficient mutant

Abstract The multi-enzyme system responsible for the biosynthesis of iturin, an antifungal lipopeptide of Bacillus subtilis , was partially purified by chromatography on different affigels. In the wild-type strain, two subunits of the iturin synthetase (ITs and ITagp) were characterized: ITs activated only l-Ser, one of the iturin amino acid components, and ITagp activated l-Asn, d-Asn, l-Gln and l-Pro, amino acids corresponding to a partial sequence of iturin. In an iturin deficient mutant, the activity of the ITagp subunit was modified.     

Effect of temperature change and aeration on the production of the antifungal peptide antibiotic iturin by Bacillus subtilis NB22 in liquid cultivation

The effects of temperature and air supply on the production of the antifungal lipopeptide antibiotic, iturin by Bacillus subtilis NB22 in a 5-d course of liquid cultivation were investigated on a flask scale. Changing the temperature from 30°C to 25, 37, 42, or 48°C for one day during the 5-d cultivation reduced the amount of iturin producted in comparison with cultivation at a constant 30°C. The temperature on the first day was notably critical. When the air supply was varied by changing the plugs of the shaking flasks, the air supply was also critical on the first day. When cells spent the first day with an inappropriate temperature or air supply, the decrease in iturin production could not be compensated for during the subsequent 4-d cultivation at an adequate temperature and air supply. Although iturin production was significant on the second and third days when the growth of the bacterium had leveled off, the initial conditions severely affected the yield of the product.     

Studies on the biosynthesis of iturin, an antibiotic of Bacillus subtilis, and a lipopeptide containing beta-hydroxy fatty acids

The biosynthesis of iturin, an antibiotic containing a beta-amino fatty acid, was studied by incubating Bacillus subtilis in the presence of various 14C-labelled precursors. Sodium acetate or palmitic acid were incorporated into the beta-amino acids of iturin. Among the alpha-amino acids (asparagine, glutamine, serine, proline and tyrosine) in the peptidic part of iturin, asparagine appears to be the best precursor. In the presence of sodium [14C]acetate or [14C]asparagine, there was a synthesis of radioactive compound (compound X) before the synthesis of radioactive iturin. Compound X contained asparagine and/or aspartic acid, glutamine and/or glutamic acid and beta-hydroxy fatty acids.     

Investigation of optimal conditions for foam separation of iturin,an antifungal peptide produced by Bacillus subtilis

An antifungal peptide, iturin, was produced by a newly-isolated Bacillus subtilis, which contains cyclic 7 d- and l-α amino acids and β-amino acids with aliphatic side chains. It consists of 6 derivatives with different length side chains. When the bacterium was cultivated in a jar fermentor, all the iturin produced was entrained in the foam. Thus, continuous separation and condensation of the product iturin is possible only by collecting the foam produced during fermentation. The controlling factors for foaming, namely the aeration rate, temperature and medium composition, especially the ratio of glucose to Polypepton, were investigated in relation to iturin production. The most effective conditions for the condensation of iturin were an aeration rate of 0.1 vvm, a temperature of 30°C, and a glucose to Polypepton ratio of 0.3–0.4. The total amount of iturin produced, and the ratio of peaks 3,4 and 5 in the iturin were maximized at 50 g/l of Polypepton by using a fermentor equipped with a basket-shaped agitation unit.     

Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis, on Micrococcus luteus.

Iturin A has an antibacterial activity on M. luteus which is strongly reduced in presence of MgCl₂. Iturin A lyses M. luteus protoplast, this lysis is enhanced by EDTA and inhibited by MgCl₂. These results suggest an action of iturin A on cytoplasmic membrane with interactions of both lipophilic and polypeptidic moieties of the antibiotic, respectively with membrane lipids and membrane polar components. Polar interactions involve the participation of mineral ions as magnesium ions have a strong inhibition effect on the activity of iturin A. The effect of iturin A on the incorporation of radio-active thymidine, uracil, isoleucine and alanine seems unspecific and is probably a consequence of the primary action on cytoplasmic membrane.     

Interactions of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis.

The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin. The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles. Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations.     

Use of soybean curd residue, okara, for the solid state substrate in the production of a lipopeptide antibiotic, iturin A, by Bacillus subtilis NB22

The possibility of the utilization of soybean curd residue, okara, for the production of a lipopeptide antibiotic, iturin A, in solid state fermentation (SSF) by Bacillus subtilis NB22 was investigated. Okara is a by-product of the tofu manufacturing process, now treated as an industrial waste and disposed of mostly by incineration. Dehydrated okara, with improved transportability and preservability, could be used as effectively as the fresh, intact okara for SSF by B. subtilis NB22 for the production of iturin A.     

Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis

Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield. The cultures were made in a synthetic medium. Several L-amino acids and various carbon sources were good substrates for the lipopeptide production. The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B. subtilis S 499.     

Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus,bacillus subtilis,and Escherichia coli

Summary A shuttle vector that could replicate in B. stearothermophilus, B. subtilis, and E. coli was constructed from B. stearothermophilus cryptic plasmid pSTK1, E. coli vector pUC19, and a thermostable kanamycin-resistance marker. This new vector was stably maintained in B. stearothermophilus at 67°C without selective pressure.     

Inhibition of some mycotoxigenic fungi by iturin A,a peptidolipid produced by Bacillus subtilis

Bacillus subtilis produces peptidolipid compounds of the iturin group that have been shown to have antifungal properties, but not all fungal species are sensitive to these compounds. In this study, the activity of iturin A, produced by B. subtilis strain B-3, was tested. Paper disks impregnated with various concentrations of iturin A were placed on agar plates seeded with conidia of toxigenic species of Fusarium, Gerlacia, Penicillium or Aspergillus. Most isolates were inhibited at iturin A concentrations as low as 4 g/disk. Penicillium italicum, P. vindicatum, A. ochraceus and A. versicolor were most strongly inhibited by the iturin whereas P. citrinum and A. parasiticus were least sensitive to iturin A.Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.     

Development of membrane lipids in the surfactin producer Bacillus subtilis

Processes occurring in the cytoplasmic membrane of the surfactin producer Bacillus subtilis were examined during a 3-d cultivation. The fatty acid composition was found to be almost stable within this interval, except for the early stationary phase when the nonbranched, mostly C(16:0) and C(18:0) (high melting fatty acids), prevailed transiently in the membrane. As for phospholipids, phosphatidylglycerol and phosphatidylethanolamine, representing 73 % of the total in the membranes of exponential cells were partly replaced by cardiolipin, which gradually rose from 5 to 28 % at the end of cultivation. In parallel, steady-state fluorescence anisotropy (r (s)) measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated a remarkable increase of r (s) DPH during the long-term cultivation and implied a continuous rigidization of the membrane interior. By contrast, the almost constant values of r (s) 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene 4-toluenesulfonate (TMA-DPH) reflected stable microviscosity of the membrane surface region. Thus, the significant increase of high melting fatty acids and cardiolipin in the cytoplasmic membrane together with the progressive rigidization of the membrane interior reflected the cell adaptation to adverse conditions.