Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.     

Biosynthesis of iturin and surfactin byBacillus subtilis: Evidence for amino acid activating enzymes

Summary Enzymes catalyzing ATP-PPi exchange reactions mediated by specific amino acids were found inB. subtilis lysates partially purified by gel permeation. The nature of these amino acids varied according to the stage of cell growth: activation of surfactin amino acids was observed during surfactin synthesis; activation of iturin amino acids was not detected at the begining of surfactin synthesis but appeared during iturin synthesis.     

Influence of the culture medium on the production of iturin A by Bacillus subtilis

The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, L-asparagine was the best substrate for the biosynthesis of iturin A; L-glutamine and L-serine were rather poor substrates while L-proline and D-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.     

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.     

Aggregational behavior of aqueous dispersions of the antifungal lipopeptide iturin A

Iturin A, a lipopeptide isolated from Bacillus subtilis, possesses a strong antifungal activity, and has been devoted to a great deal of attention. Since iturin is an amphiphilic compound with a great propensity to self-associate in solution as well as inside the membrane, the question arises to whether its aggregational behavior is dependent on the concentration of the lipopeptide. In order to test this, the ability of iturin suspensions to encapsulate water-soluble molecules has been examined. Iturin was dispersed at different concentrations above its critical micellar concentration, in a buffer containing the water-soluble dye 5,6-carboxyfluorescein. For iturin A micelles, a Stokes radius of 1.3 nm and an aggregational number of 7 was obtained. The results shown in this work clearly demonstrate that iturin dispersions in water, at concentrations of 0.7, 1.4 and 3 mM, i.e. far above the critical micellar concentration (40 microM), are capable of encapsulating carboxyfluorescein, probably by adopting a type of aggregate different from the micelle. Negative-staining electron microscopy shows the presence of vesicles with an average size of 150 nm. By using (14)C-iturin, it is shown that, at 3 mM concentration, 40 % of the iturin molecules adopt this vesicular state. It is proposed that iturin molecules form a fully interdigitated bilayer, where each hydrocarbon tail span the entire hydrocarbon width of the bilayer, resulting in multilamellar vesicles capable of encapsulating an aqueous compartment. The possible implications of these results to the membrane destabilizing effect of iturin A, are discussed according to the dynamic cone-shape of the iturin molecule.     

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.     

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.     

The unconventional adverse effects of fungal pretreatment on iturin A fermentation by Bacillus amyloliquefaciens CX-20

Fungal pretreatment is the most common strategy for improving the conversion of rapeseed meal (RSM) into value-added microbial products. It was demonstrated that Bacillus amyloliquefaciens CX-20 could directly use RSM as the sole source of all nutrients except the carbon source for iturin A fermentation with high productivity. However, whether fungal pretreatment has an impact on iturin A production is still unknown. In this study, the effects of fungal pretreatment and direct bio-utilization of RSM for iturin A fermentation were comparatively analysed through screening suitable fungal species, and evaluating the relationships between iturin A production and the composition of solid fermented RSM and liquid hydrolysates. Three main unconventional adverse effects were identified. (1) Solid-state fermentation by fungi resulted in a decrease of the total nitrogen for B. amyloliquefaciens CX-20 growth and metabolism, which caused nitrogen waste from RSM. (2) The released free ammonium nitrogen in liquid hydrolysates by fungal pretreatment led to the reduction of iturin A. (3) The insoluble precipitates of hydrolysates, which were mostly ignored and wasted in previous studies, were found to have beneficial effects on producing iturin A. In conclusion, our study verifies the unconventional adverse effects of fungal pretreatment on iturin A production by B. amyloliquefaciens CX-20 compared with direct bio-utilization of RSM.     

Specific inhibition of iturin biosynthesis by cerulenin

Cerulenin, an inhibitor of fatty acid synthesis, inhibits the biosynthesis of iturin by Bacillus subtilis. With a cerulenin concentration of 2 micrograms/mL, 50% inhibition was achieved. At this concentration, cerulenin does not affect growth or total protein synthesis but does inhibit the incorporation of sodium [14C]acetate, [14C]myristic acid, and [14C]asparagine into iturin. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, the inhibition of iturin and beta-amino acid syntheses by cerulenin is discussed in relation with lipid synthesis.     

Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids

Iturin A, extracted from the culture media of Bacillus subtilis, is an antifungal lipopeptide, the peptide cycle of which includes a D-Tyr residue in position 2. The antibiotic strength of iturin A is related to a change in the permeability of the membrane cells which leads to a leakage of K+ from the intracellular medium. Methylation of the D-Tyr residue dramatically decreases the biological activity of iturin A. Using the intrinsic fluorescence of D-Tyr we have shown that both iturin A and O-methyl-tyrosine iturin A enter the lipid membranes. When dimyristoylphosphatidylcholine vesicles contain iturin A we observe a change in the order degree of the lipid phase and an increase in the transition temperature. The methylated derivative has no effect. Two model membranes have been used to study the permeability changes induced by iturin A and O-methyltyrosine iturin A. Studying ionic permeability we have found that the conductance of a planar lipid membrane increases very much less when the lipopeptide is methylated. On the other hand, the release of carboxyfluorescein trapped in lipid vesicles is less upon addition of O-methyltyrosine-iturin A. We conclude that the Tyr residue of the peptide cycle plays a role in determining the interactions of iturin A with lipid membrane.     

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.     

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

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 defined as second stage production.     

Interactions of the lipopeptide antifungal iturin A with lipids in mixed monolayers

The miscibility and the interactions of the antifungal lipopeptide iturin A with lipids, DMPC and cholesterol, are studied in monolayers at the air/water interface and a comparison of the respective behaviour of iturin A and the biologically inactive methylated derivative MeTyr-iturin A is made. Each lipopeptide is miscible with anyone of the lipids. This behaviour is revealed by the dependence of the transition pressure upon composition and by deviations from the additivity rule of the mean molecular area. The thermodynamic properties of the mixed systems are studied by the method of Goodrich. The mixed monolayers are always more stable than the two separate components, subsequently there are interactions between the components. However, the excess free energy of mixing delta Gexm is positive for the iturin A/DMPC system which is an indication that the interactions between lipopeptide and lipid molecules are weaker than the interactions between the pure components themselves. This is compatible with the presence of self-associated lipopeptide molecules. However, delta Gexm is highly negative for the iturin A/cholesterol system giving evidence of the formation of a specific complex between iturin A and cholesterol which is not the case with the methylated derivative. These data are analysed in connection with previous results concerning the pore-forming properties of these lipopeptides and the lack of biological activity of MeTyr-iturin A.     

新型抗菌肽——表面活性素、伊枯草菌素和丰原素

表面活性素（surfactin）、伊枯草菌素（iturin）和丰原素（fengycin）是一类主要由革兰阳性芽胞杆菌通过非核糖体合成途径产生的抗菌肽,一般是由1个β-羟基脂肪酸与7～10个氨基酸肽链以酰胺键连接而成的环肽,具有抗细菌、抗真菌、抗病毒、抗肿瘤等生物活性,在医疗方面具有良好的应用前景。目前,人们对这3种新型抗菌肽在医药领域中的研究进展所知甚少,故本文对其发现历史、结构特点、作用机制、生物合成和应用价值进行阐述,为后续研究提供借鉴。     

Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A

The bacterial lipopeptide iturin A is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Hemolysis takes place at iturin concentrations below its critical micellar concentration. Relative kinetics determinations clearly show that K(+) leakage occurs prior to hemoglobin release. Furthermore, hemolysis can be prevented by addition to the outer solution of osmotic protectants of appropriate size. Altogether these results indicate that iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 A. Iturin A is capable of inducing leakage of an aqueous fluorescent probe trapped in human erythrocyte ghosts, but not in large unilamellar liposomes made of various lipid compositions. The different permeabilizing effects of iturin A on model and biological membranes are discussed on the light of the presented results.     

Quantification of the antifungal lipopeptide iturin A by high performance liquid chromatography coupled with aqueous two-phase extraction

Iturin A, a powerful antifungal surfactant, is a kind of bacterial lipopeptide produced by Bacillus strains. This study addresses the use of an aqueous two-phase system (ATPS) using ethanol/ammonium sulfate to extract iturin A from Bacillus amyloliquefaciens NJN-6 fermentation broth and the quantification of iturin A by HPLC. Baseline separation of iturin A homologues was performed using an RP-C(18) column with a mixture of water and acetonitrile. The results showed that the correlation coefficient between integral area and concentration was 0.9961 within the range of 20-140 mg/l. The RSD of the retention time and the peak area were 1.29% and 1.45%, respectively. The effects of some operating parameters in ATPS, e.g., pH, temperature and centrifugation time, were also studied. This method can be successfully used for the rapid quantification of iturin A.     

Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A

The bacterial lipopeptide iturin A is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Hemolysis takes place at iturin concentrations below its critical micellar concentration. Relative kinetics determinations clearly show that K⁺ leakage occurs prior to hemoglobin release. Furthermore, hemolysis can be prevented by addition to the outer solution of osmotic protectants of appropriate size. Altogether these results indicate that iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 Å. Iturin A is capable of inducing leakage of an aqueous fluorescent probe trapped in human erythrocyte ghosts, but not in large unilamellar liposomes made of various lipid compositions. The different permeabilizing effects of iturin A on model and biological membranes are discussed on the light of the presented results.     

The influence of process parameters in production of lipopeptide iturin A using aerated packed bed bioreactors in solid-state fermentation

The strain Bacillus iso 1 co-produces the lipopeptide iturin A and biopolymer poly-γ-glutamic acid (γ-PGA) in solid-state fermentation of substrate consisting of soybean meal, wheat bran with rice husks as an inert support. The effects of pressure drop, oxygen consumption, medium permeability and temperature profile were studied in an aerated packed bed bioreactor to produce iturin A, diameter of which was 50 mm and bed height 300 mm. The highest concentrations of iturin A and γ-PGA were 5.58 and 3.58 g/kg-dry substrate, respectively, at 0.4 L/min after 96 h of fermentation. The low oxygen uptake rates, being 23.34 and 22.56 mg O₂/kg-dry solid substrate for each air flow rate tested generated 5.75 W/kg-dry substrate that increased the fermentation temperature at 3.7 °C. The highest pressure drop was 561 Pa/m at 0.8 L/min in 24 h. This is the highest concentration of iturin A produced to date in an aerated packed bed bioreactor in solid-state fermentation. The results can be useful to design strategies to scale-up process of iturin A in aerated packed bed bioreactors. Low concentration of γ-PGA affected seriously pressure drop, decreasing the viability of the process due to generation of huge pressure gradients with volumetric air flow rates. Also, the low oxygenation favored the iturin A production due to the reduction of free void by γ-PGA production, and finally, the low oxygen consumption generated low metabolic heat. The results show that it must control the pressure gradients to scale-up the process of iturin A production.     

Influence of iturin A on mycelial weight and aflatoxin production byAspergillus flavus andAspergillus parasiticus in shake culture

Iturin A, a peptidolipid produced byBacillus subtilis, inhibits growth of a large number of fungi. In this study, the effects of iturin A were evaluated on nine isolates ofA. flavus and seven isolates ofA. parasiticus in liquid shake culture. The mycelial dry weight of theA. flavus isolates was not significantly influenced by iturin A, however, there was a significant reduction in mycelial dry weight for two of theA. parasiticus isolates. Aflatoxin production was significantly reduced in five of theA. flavus isolates and three of the six aflatoxigenicA. parasiticus isolates. For the other seven isolates, aflatoxin levels were either unchanged or significantly increased in the presence of iturin A. These results indicate that iturin A does not consistently reduce growth or aflatoxin production of these fungi in pure culture.