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.     

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.     

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.     

Characterization of the surfactin synthetase isolated from the Bacillus subtilis strain producing iturin

Summary The lipopeptides, surfactin and iturin, are co-produced by B. subtilis. In this work, the three subunits of surfactin synthetase have been characterized by affinity chromatography on affigel columns where the ligand is one of the amino acid components of surfactin.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

DNA-relaxing enzyme from Micrococcus luteus.

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.     

Structure of iturine A, a peptidolipid antibiotic from Bacillus subtilis.

A mixture of iturines extracted from Bacillus subtilis gave, on column chromatography, iturine A, iturine B, and iturine C. Iturine A has the entire antifungal activity. It is a mixture of two homologous peptidolipids C48,H74N12O14 and C49H76N12O14 (mp 177 degrees C, [alpha]D-1.7 degrees in methanol (c 0.05 g/mL); mol wt 1042 and 1056). The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid. The peptide moiety contains 7 mol of amino acids: D-Asn2, L-Asn, L-Gln, L-Pro, L-Ser, and D-Tyr. A cyclic structure for iturine A with the serine residue linked to the fatty amino acids through a peptide bond has been domonstrated. By mild HCl hydrolysis, lipid-soluble and water-soluble peptides were obtained. They were analyzed by chemical methods and by mass spectrometry. Permethylated and perdeuteriomethylated derivatives of iturine A were also subjected to mass spectrometric analysis. Both chemical analysis and mass spectrometry led to the cyclic structure I for iturine A.     

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.     

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.     

Mechanism of action of Micrococcus luteus gamma-endonuclease

Micrococcus luteus extracts contain gamma-endonuclease, a Mg2+-independent endonuclease that cleaves gamma-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. gamma-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. gamma-Endonuclease cleaves DNA containing thymine glycols or apurinic sites immediately 3' to the damaged or missing base. Cleavage results in a 5'-phosphate terminus and a 3' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of gamma-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.     

Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.     

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.     

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.     

藤黄微球菌纤溶酶基因在枯草芽孢杆菌WB600中的表达

MLFE(Micrococcus luteus fibrinolytic enzyme)是由一株新的纤溶酶产生菌藤黄微球菌ML909分泌的胞外纤溶酶。从一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens)DC-4中克隆a-淀粉酶基因的启动子－信号肽序列, 将其与mlfe基因(GenBank, 登录号为EU232121)的成熟肽编码序列相融合, 构建成融和基因amymlfe。将该基因克隆到大肠杆菌－枯草芽孢杆菌穿梭质粒pSUGV4上, 构建成表达质粒pSU-AmyMLFE, 再将其转化     

Interactions between bacterial membranes and peptidolipids: Lysis of Micrococcus luteus protoplasts by derivatives of peptidolipidic antibiotics from Bacillus subtilis

The lysis of protoplasts of Micrococcus luteus has been tested with various derivatives of three peptidolipidic antibiotics: iturin A, mycosubtilin and bacillomycin L. The lytic activity is dependent to the nature of the substituting group and to the position of the substituted aminoacid residue. The acetylation of OH groups leads to a decrease of the lytic activity of the natural antibiotics. The methylation of aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity. The methylation of the tyrosyl residue enhances the lytic activities of iturin A and of bacillomycin L-dimethyl ester and reduces that of mycosubtilin.Correlations between the structures of derivatives and their lytic action on M. luteus protoplasts are discussed.     

Gamma endonuclease of Micrococcus luteus: action on irradiated DNA

Gamma endonuclease is a Mg2+-independent enzyme of Micrococcus luteus that recognizes and cleaves DNA at a variety of altered pyrimidines produced by ionizing radiation. The production of enzyme-recognizable sites (ERS) by ionizing radiation under different irradiation conditions was measured. Ionizing radiation produced the greatest number of ERS when irradiations were performed under anoxic conditions in the presence of the free radical scavenger KI. Since dihydrothymine is a major pyrimidine lesion produced in DNA during anoxic irradiation, the ability of gamma endonuclease to excise this lesion was assessed. Dihydrothymine was released from DNA irradiated under anoxic conditions in a radiation dose-dependent manner, consistent with gamma endonuclease's known DNA glycosylase activity. Gamma endonuclease was also shown to cleave heavily uv-irradiated DNA. When the sequence specificity of gamma-endonuclease cleavage was studied using uv-irradiated DNA, cleavage was seen specifically at cytosines. The identity of this enzyme-recognizable cytosine photoproduct is not known.