ABSTRACT
New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.
Subject(s)
Amino Acids/chemistry , Asparagine/analogs & derivatives , Asparagine/chemistry , Bacillus/chemistry , Surface-Active Agents/chemistry , Amino Acids/isolation & purification , Chromatography, High Pressure Liquid , Fatty Acids/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Surface-Active Agents/isolation & purification , Time FactorsABSTRACT
The lipopeptide lichenysin (cyclo-[L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid]) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis. The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin. This local variation causes significant changes in the properties of the molecule compared to surfactin. Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM. Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively. This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange. Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane. Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules. Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures. Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved.
Subject(s)
Bacterial Proteins/chemistry , Cations , Chelating Agents/pharmacology , Lipoproteins/chemistry , Peptides, Cyclic/chemistry , Surface-Active Agents/chemistry , Calcium/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Erythrocytes/metabolism , Humans , Kinetics , Lipopeptides , Lipoproteins/pharmacology , Magnesium/metabolism , Peptides/chemistry , Peptides, Cyclic/pharmacology , Protein Binding , Surface Tension , Surface-Active Agents/pharmacologyABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/chemistry , Peptides , Anti-Bacterial Agents/pharmacology , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Bacillus subtilis , Peptides, Cyclic , Protein BindingABSTRACT
The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m-1 to 27 mN m-1 at a concentration as low as 20 microM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a beta-hydroxy fatty acid with 13-15 C atoms. In solution, the molecule exhibits a characteristic "horse saddle" conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4'-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries.
Subject(s)
Bacterial Proteins , Peptides, Cyclic , Surface-Active Agents , Bacillus/genetics , Bacillus/growth & development , Bacillus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Fermentation , Lipopeptides , Peptide Synthases/genetics , Peptide Synthases/metabolism , Protein Structure, Tertiary , Structure-Activity Relationship , Surface-Active Agents/chemistry , Surface-Active Agents/metabolismABSTRACT
A series of 9 lactonic lipopeptide biosurfactants was isolated from Bacillus licheniformis IM 1307 as representatives of the lichenysin group and we propose to name them lichenysins G. They were recovered from the culture medium as complex mixtures of molecules having different peptide sequences and different structures of beta-hydroxy fatty acids. Their separation was achieved by a reversed-phase HPLC method leading to eight well-separated compounds. The complete structure of individual isoforms was proposed following the results of amino acid and fatty acid analysis, LSI-MS and 2D NMR spectroscopies. Compared to surfactin, lichenysins G are at least 10 fold more efficient biosurfactants.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Bacillus/chemistry , Lipoproteins/isolation & purification , Peptides, Cyclic/isolation & purification , Peptides , Surface-Active Agents/isolation & purification , Amino Acids/analysis , Amino Acids/chemistry , Anti-Bacterial Agents/chemistry , Antibiotics, Antineoplastic/chemistry , Bacterial Proteins/chemistry , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Fatty Acids/analysis , Fatty Acids/chemistry , Gas Chromatography-Mass Spectrometry , Lactones/chemistry , Lactones/isolation & purification , Lipopeptides , Lipoproteins/chemistry , Magnetic Resonance Spectroscopy , Peptides, Cyclic/chemistry , Surface Tension , Surface-Active Agents/chemistryABSTRACT
Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and posses several other interesting biological activities. By means of differential scanning calorimetry and X-ray diffraction the effect of surfactin on the phase transition properties of bilayers composed of different phospholipids, including lipids forming hexagonal-HII phases, has been studied. The interactions of surfactin with phosphatidylcholine and phosphatidylglycerol seem to be optimal in the case of myristoyl acyl chains, which have a similar length to the surfactin hydrocarbon tail. Data are shown that support formation of complexes of surfactin with phospholipids. The ionized form of surfactin seems to be more deeply inserted into negatively charged bilayers when Ca2+ is present, also supporting the formation of surfactin-Ca2+ complexes. In mixtures with dielaidoylphosphatidylethanolamine, a hexagonal-HII phase forming lipid, surfactin displays a bilayer stabilizing effect. Our results are compatible with the marked amphiphilic nature of surfactin and may contribute to explain some of its interesting biological actions; for instance the formation of ion-conducting pores in membranes.
Subject(s)
Bacterial Proteins/chemistry , Lipid Bilayers/chemistry , Peptides, Cyclic , Phospholipids/chemistry , Calorimetry, Differential Scanning , Lipopeptides , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Phosphatidylglycerols/chemistry , Temperature , X-Ray DiffractionABSTRACT
The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Lipoproteins/chemistry , Lipoproteins/isolation & purification , Peptides, Cyclic , Amino Acids/analysis , Bacillus subtilis , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Isoleucine/chemistry , Lipopeptides , Lipoproteins/biosynthesis , Magnetic Resonance Spectroscopy , Mass Spectrometry , Structure-Activity Relationship , Surface TensionABSTRACT
The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Candida albicans/drug effects , Peptides , Saccharomyces cerevisiae/drug effects , Candida albicans/cytology , Candida albicans/ultrastructure , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Freeze Fracturing , Microbial Sensitivity Tests , Microscopy, Electron , Microscopy, Electron, Scanning , Peptides, Cyclic , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/ultrastructure , Time FactorsABSTRACT
When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L-valine residue by L-alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.
Subject(s)
Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , Peptides, Cyclic , Alanine/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Chromatography, Gas , Chromatography, Thin Layer , Culture Media , Hydrolysis , Lipopeptides , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Stereoisomerism , Structure-Activity Relationship , Surface Tension , Surface-Active Agents/chemistry , Valine/chemistryABSTRACT
Iturins are a family of lipopeptides extracted from the culture media of various strains of Bacillus subtilis. These amphiphilic compounds are characterized by a peptide ring of seven amino acid residues including an invariable D-Tyr2, with the constant chiral sequence LDDLLDL closed by a C14-C17 aliphatic beta-amino acid. They exhibit strong antifungal activities against a wide variety of pathogenic yeasts and fungi but their antibacterial activities are restricted to some bacteria such as Micrococcus luteus. The biological activity of the iturin lipopeptides is modulated by the primary structure of the peptide cycle as illustrated by the methylation of the D-Tyr2 residue which dramatically decreases the activity or by the inversion of the two adjacent Ser6-Asn7 residues which makes mycosubtilin more active than iturin A. The antifungal activity is related to the interaction of the iturin lipopeptides with the cytoplasmic membrane of target cells, the K+ permeability of which is greatly increased. The ability of iturin compounds to increase the membrane cell permeability is due to the formation of ion-conducting pores, the characteristics of which depend both on the lipid composition of the membrane and on the structure of the peptide cycle. From monolayer experiments it has been suggested that these ionic pores are the consequence of the presence of aggregates (lipopeptide aggregates or lipopeptide/phospholipid complex aggregates) in the phospholipid membrane. It has also been shown that, when active, iturins interact strongly with sterols, forming lipopeptide/cholesterol complexes. Therefore, the biologically efficient structure might be a ternary structure: iturin/phospholipid/sterol.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Peptides , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Cell Membrane Permeability/drug effects , Humans , Lipoproteins/pharmacology , Membrane Potentials , Molecular Sequence Data , Peptides, CyclicABSTRACT
Iturin A, a lipopeptide from Bacillus subtilis, induced morphological modifications of human erythrocytes and a concomitant release of exovesicles. The modifications depended on the lipopeptide concentration and on the incubation time. Crenation of cells occurred while invaginations appeared when the incubation time was increased. This suggested that iturin A was gradually translocated from the outer layer into the inner half of the bilayer. For one lipopeptide concentration, heterogeneity in the form of the erythrocytes was observed which could result from heterogeneity in the self-association of iturin A. The lipid composition of exovesicles was markedly different from that of the native erythrocytes; they were enriched in cholesterol, sphingomyelin and phosphatidylcholine. Freeze-fracture showed that the ultrastructure of the protoplasmic face of the membrane was not modified by iturin A but, when erythrocytes were prefixed with glutaraldehyde prior to iturin A treatment, irregular smooth protuberances appeared with a decrease of intramembranous particles density. These protuberances which covered the complete area of the cell may have been complex associations of iturin A, phospholipids and cholesterol.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacillus subtilis/chemistry , Erythrocyte Membrane/drug effects , Erythrocytes/drug effects , Peptides , Cell Membrane Permeability/drug effects , Cholesterol/analysis , Erythrocyte Membrane/metabolism , Erythrocyte Membrane/ultrastructure , Erythrocytes/chemistry , Erythrocytes/ultrastructure , Filipin/metabolism , Humans , Membrane Proteins/analysis , Microsomes/chemistry , Microsomes/ultrastructure , Peptides, Cyclic , Phospholipids/analysisABSTRACT
Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.
Subject(s)
Anti-Bacterial Agents , Antibiotics, Antineoplastic/pharmacology , Antifungal Agents/pharmacology , Bacterial Proteins/pharmacology , Hemolysis/drug effects , Peptides , Adsorption , Amino Acid Sequence , Antibiotics, Antineoplastic/chemistry , Antifungal Agents/chemistry , Bacterial Proteins/chemistry , Drug Interactions , Drug Synergism , Erythrocytes/drug effects , Humans , Lipopeptides , Molecular Sequence Data , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacologyABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Antifungal Agents/pharmacology , Bacterial Proteins/pharmacology , Erythrocyte Membrane/drug effects , Peptides , Saccharomyces cerevisiae/drug effects , Amino Acid Sequence , Antifungal Agents/chemistry , Antifungal Agents/metabolism , Bacillus subtilis , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Drug Synergism , Erythrocyte Membrane/metabolism , Hemolysis , Humans , Lipopeptides , Molecular Sequence Data , Peptides, Cyclic/chemistry , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Saccharomyces cerevisiae/cytology , Spectrometry, Fluorescence , Surface TensionABSTRACT
Reinvestigation of surfactin, a previously studied peptidolipid surfactant from Bacillus subtilis, by fast-atom-bombardment mass spectrometry and 1H-NMR spectroscopy, as well as by chemical methods, revealed the presence of a closely related second constituent. This new compound, [Val7]surfactin, differs from the known surfactin by the C-terminal amino acid residue which is valine instead of leucine.
Subject(s)
Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , Genetic Variation , Peptides, Cyclic , Valine/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Lipopeptides , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Antifungal Agents/biosynthesis , Bacillus subtilis/metabolism , Bacterial Proteins/biosynthesis , Peptides , Surface-Active Agents/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Antifungal Agents/isolation & purification , Bacterial Proteins/isolation & purification , Bacterial Proteins/pharmacology , Carbohydrate Metabolism , Chromatography, Thin Layer , Lipopeptides , Lipoproteins/biosynthesis , Lipoproteins/isolation & purification , Lipoproteins/pharmacology , Molecular Sequence Data , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Surface-Active Agents/isolation & purificationABSTRACT
The binding of the membrane-active lipopeptide antibiotic iturin A to yeast cells was studied using radioactive iturin A. Saccharomyces cerevisiae had a maximum binding capacity of 5.6 x 10(9) molecules per single cell. The Scatchard plot of binding showed a biphasic profile, with a lower dissociation constant for small concentrations of iturin A. The break of slope at 30 microM iturin A corresponds to the micellization of antibiotic in solution. The binding is also dependent on the nature of the sterol present in the membrane. A mutant yeast strain with a membrane containing cholesterol instead of ergosterol showed the highest affinity for iturin A and the highest sensitivity to this antibiotic, as measured by K+ ion release. In contrast the presence of stigmasterol increased the resistance of the cells to iturin A.
Subject(s)
Anti-Bacterial Agents , Antifungal Agents/pharmacology , Cholesterol/metabolism , Peptides , Phytosterols/metabolism , Saccharomyces cerevisiae/drug effects , Stigmasterol/metabolism , Cell Membrane/analysis , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Drug Resistance, Microbial , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Potassium/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Spheroplasts/metabolismABSTRACT
The action of iturin A on non-growing cells of Saccharomyces cerevisiae was tested. This antibiotic gave important modifications in the membrane permeability which permitted nucleotides, proteins, polysaccharides and lipids to escape from cells. The lipid content of cells was strongly disturbed; the level of phospholipids, essentially phosphatidylcholine, decreased while the level of fatty acids increased. A part of these fatty acids were extruded from yeast cells. The role of iturin A in these modifications was discussed.
Subject(s)
Anti-Bacterial Agents , Antifungal Agents/pharmacokinetics , Cell Membrane Permeability/drug effects , Peptides , Saccharomyces cerevisiae/drug effects , Peptides, Cyclic/pharmacokinetics , Phospholipids/analysisABSTRACT
A comparison has been made between the pore-forming properties of the antibiotic lipopeptide iturin A and a derivative methylated on the tyrosine residue which shows a restricted biological activity. It is shown that this derivative increases the ion permeability of planar lipid membranes as does iturin A. Nevertheless, the global conductance of the doped membrane is very much lower at the same lipopeptide/phospholipid ratio and the ion selectivity is inverted (PK/PCl = 6 instead of 0.6 with iturin A). The characteristics of the induced conducting pores are also rather different. This suggests an important role of the D-Tyr2 residue, present in all the compounds of the iturin family, both in the biological and in the pore-forming properties of iturin A.
Subject(s)
Anti-Bacterial Agents , Peptides , Tyrosine , Cholesterol , Liposomes , Membrane Potentials , Methylation , Models, Biological , Peptides, Cyclic , PhosphatidylcholinesABSTRACT
The structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis, was revised by FAB mass spectrometry, 2D NMR spectrometry and also by Edman degradation of the peptide resulting from the N-bromosuccinimide reaction. Four homologous beta-amino acid components were identified by capillary gas chromatography. The cyclopeptide mycosubtilin consists of seven alpha-amino acids in an LDDLLDL sequence closed by a beta-amino acid linkage similar to that found in other antibiotics of the iturin group.