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.     

Interactions of antibiotics of the iturin group with human erythrocytes

The peptidolipid antibiotics, iturin A and bacillomycin L have a disrupting effect on erythrocyte membrane leading to a simultaneous release of K+ ions and hemoglobin. The formation of ghosts is accompanied by a partial solubilisation of lipid components. Binding experiments with radioactive antibiotics show that about 7 X 10(7) molecules of iturin A and 4 X 10(7) molecules of bacillomycin L are bound to one erythrocyte at the concentration giving 100% hemolysis. This concentration is reduced by about 20% after treatment of erythrocytes by phospholipase A2. Scatchard plots show that the affinity for erythrocyte membrane is higher with bacillomycin L than with iturin A. This difference is probably correlated to the differences in their peptide moieties. The substitution of tyrosyl residue leads to a ten-fold increase of the concentrations giving 100% hemolysis, probably due to a low distribution coefficient of derivatives between the membrane and the solvent. This result and the humped Scatchard curves obtained with both antibiotics may be related to the self-association of the compounds in aqueous solutions.     

Action of peptidolipidic antibiotics of the iturin group on erythrocytes: Effect of some lipids on hemolysis

Iturin A, bacillomycin L and bacillomycin L dimethyl ester have a strong lytic activity upon human erythrocytes while iturin C is totally inactive. The hemolytic action of the antibiotics is inhibited by free cholesterol as well as by cholesterol included in mixed liposomes of phosphatidylcholine-cholesterol and to a lesser extent by phosphatidylcholine liposomes. This inhibition is the result of an interaction between the antibiotic and added lipids which diminishes the concentration of free antibiotic available to lyse erythrocytes. The inhibitory effect of liposomes on hemolysis demonstrates the affinity of the antibiotic for artificial membranes, especially those containing cholesterol.     

Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D

Bacillus subtilis AU195 produces bacillomycin D, a cyclic lipopeptide that is an inhibitor of the aflatoxin producing fungus Aspergillus flavus. Sequence analysis of the bacillomycin D operon revealed four ORFs with the structural organization of the peptide synthetases. Disruption of ORF 2, which links the amino acid moiety to the b-amino fatty acid, resulted in the loss of antifungal activity. By comparing the sequence of bacillomycin D, iturin A and mycosubtilin operons, our results showed that intergenic module replacement have occurred between B. subtilis lipopeptide synthetases including the iturin family and the plipastatin and fengycin family.     

Influence of divalent ions on the solubility of iturin and bacillomycin L, antifungal peptidolipids of Bacillus subtilis

When calcium chloride was added to the culture medium of strains producing iturin or bacillomycin L, the antibiotic, normally excreted in the supernatant of the culture medium, was found in the cell pellet. This apparent inhibition of the antibiotic excretion was studied and it was demonstrated that CaCl2 precipitated the antibiotic after its excretion in the medium. The ability of various chloride salts to precipitate iturin and bacillomycin L was tested and the most effective salts were CaCl2 and MnCl2. Comparison of the compounds obtained by CaCl2 precipitation and by acid precipitation showed that, in the latter case, major antibiotics were accompanied by minor congeners resulting from modifications of genuine antibiotics.     

Effect of gramicidin S and its derivatives on protoplasts of Bacillus subtilis

The lysis of Bacillus subtilis protoplasts by gramicidin S, a membrane active antibiotic, and its derivatives was studied according to free amino groups of the ornithine residue. The initial antibiotic and guanylgramicidin , a positive charge-preserving derivative, had a high lytic activity. Succinylgramicidin , a gramicidin S derivative with acid properties, and carbomoylgramicidin , a neutral derivative, actively lysed B. subtilis protoplasts suspended in 1/15 M phosphate buffer solution with sucrose . No lytic activity of succinylgramicidin was observed with respect to B. subtilis protoplasts suspended in an aqueous solution of sucrose. Comparative study on the sensitivity of the protoplasts of Micrococcus lysodeikticus, B. megaterium and B. subtilis to the lytic action of gramicidin S and its derivatives showed in the main a similar character of their interaction with the membranes of the protoplasts of the taxonomically close species (B. megaterium and B. subtilis). It is likely that the specificity of the action of the above substances on the protoplasts of M. lysodeikticus, i. e. a complicated character of the dependence of the lytic action of gramicidin S on its concentration, manifestation of the lytic activity of the neutral and acid derivatives in the presence of phosphates or other salts and in sucrose aqueous solution was mainly defined by the properties of the micrococcal membranes.     

小麦赤霉病原菌拮抗菌Bacillus amyloliquefaciens 7M1产抗菌素的研究

【目的】从小麦根际土壤分离鉴定一株赤霉病拮抗菌,对该菌产的抗菌素进行生物学性质研究、种类鉴定和抑菌实验。【方法】利用牛津杯法和光照培养箱实验对其抑菌活性进行测定,通过16S r RNA基因序列分析对目标菌株的种属进行初步鉴定,根据抗菌素相关基因进行PCR扩增和测序,利用在线软件Pro Param tool和TMHMM对编码蛋白进行生物信息学分析。【结果】7M1菌体和抗菌素对禾谷镰刀菌的抑菌圈直径分别为16.33±0.13 mm和15.43±0.21 mm,16S r RNA基因序列分析结果显示其为芽孢杆菌,并与解淀粉芽孢杆菌具有较近的亲缘关系,菌株7M1抗菌素对小麦赤霉病的温室防治效果为76.41%,而且热稳定性好,可被蛋白酶K、胰蛋白酶、胃蛋白酶降解,在p H 5.0-10.0有较好的抑菌活性,但是紫外线辐射会降低其抑菌活性。菌株7M1含有bac AB、itu C、bam D 3种基因,通过与Gen Bank中相关的抗菌素基因进行比对,发现其编码的氨基酸序列与Gen Bank库中的芽孢杆菌溶素、伊枯草菌素和杆菌抗霉素D等抗菌素的相似性达到99%。bac AB编码蛋白和itu C编码蛋白是稳定蛋白,bam D编码蛋白是不稳定蛋白,此外,3种基因的编码产物不具有明显的跨膜结构。【结论】从该菌发酵液提取的抗菌素有很好的抗禾谷镰刀菌活性而且性质稳定,因而在小麦赤霉病的生物防治中具有潜在的应用价值。     

Production of human lactoferrin in animal milk

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.     

The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.     

Action of mycosubtilin, an antifungal antibiotic of Bacillus subtilis, on the cell membrane of Saccharomyces cerevisiae

Mycosubtilin, an antibiotic of the iturin group, inhibits the growth of Saccharomyces cerevisiae by a fungicidal action. Increasing concentrations of mycosubtilin decrease the incorporation of radioactive precursors into proteins, RNA and polysaccharides without specificity. Yeast spheroplasts are lysed by mycosubtilin. Its action on the cytoplasmic membrane induces important modifications of the membrane permeability: nucleotides, proteins and lipids are released from the cells. These releases increase with increasing concentrations of mycosubtilin.     

Action of different types of gramicidin S derivatives on bacterial cells and protoplasts

The work was concerned with studying the effect of gramicidin S derivatives with modified free amino groups of ornithine residues on bacterial cells and protoplasts. The substitution of the amino groups with neutral or carboxyl-containing groups eliminated or sharply decreased the antibacterial activity of gramicidin S, its binding to the cells, and the ability to change the permeability of the cytoplasmic membranes of the intact cells. However, the neutral derivatives and the derivative with acidic properties showed a considerable lytic activity when they were incubated with the protoplasts of Micrococcus lysodeikticus, Bacillus megaterium and Bacillus subtilis. Hence, these compounds preserved a certain membranotropic level. Those gramicidin S derivatives with modified ornithine amino groups which possessed basic properties were similar to gramicidin S in the antibiotic activity, the modified permeability of the membranes, the ability to bind with the cells, and the lytic action on the protoplasts.     

Effect of salts on the lytic activity of gramicidin S and its derivatives

Potassium and sodium chlorides, sulfates, acetates and phosphates activated the lytic action of gramicidin S and its derivatives on protoplasts of M. lysodeikticus. The derivatives used were positively charged and neutral by the free amino groups in the ornithine moieties. The salts had no effect on lysis of the bacillar protoplasts by gramicidin S and its positively charged derivatives. The lytic effect of the neutral derivative on the bacillar protoplasts markedly increased in the presence of the salts, activation of the lysis by the phosphates being more pronounced than that by the other salts. Increased membrane activity of gramicidin S in the presence of the salts was not connected with association of the substance molecules in solution. Probably it was due to increased destruction of the membranes at the account of activated detergent effect of the antibiotic and its derivatives.     

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.     

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.     

Effect of polyene antibiotics on bacterial protoplasts

The carbonyl-conjugated pentaenes flavofungin, nigrofungin and flavopentin exhibit considerable lytic activity toward Micrococcus lysodeikticus and Bacillus megaterium protoplasts. The antibiotics at concentrations of 5 to 14 microgram/ml cause lysis of 50% of the protoplasts within 15 min of their incubation. The antibiotics inhibit the activity of NADH oxidase and malate oxidase by 50% in the lysates of Micrococcus lysodeikticus and Bacillus megaterium protoplasts at concentrations of 30 to 50 microgram/ml; preincubation of the lysates with the antibiotics intensify the inhibiting action of the polyenes. Growth of the bacteria is inhibited when the minimal concentration of the polyenes is 75 to 100 microgram/ml. Interaction of the polyenes with bacterial membranes lacking sterols indicates that resistance of at least some bacteria to polyenes is caused by impermeability of the cell wall for these substances rather than by the absence of sterols in the membranes.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

Characterisation of antagonistic Bacillus and Pseudomonas strains for biocontrol potential and suppression of damping‐off and root rot diseases

Novel strains of rhizobacteria, Pseudomonas fluorescens Pf 9A‐14, Pseudomonas sp. Psp. 8D‐45 and Bacillus subtilis Bs 8B‐1, showed broad‐spectrum antagonistic activity and provided suppression of Pythium damping‐off and root rot of cucumber. Their biocontrol potential was further investigated for suppression of additional seedling diseases of cucumber (Phytophthora capsici) and radish (Rhizoctonia solani). Bacterial strains were also characterised for production of antibiotics, metabolites, volatiles, phytohormones and lytic enzymes. Seed and pre‐plant applications of all three antagonistic bacteria as cell suspension and talc or irradiated peat formulations to the infested potting mix provided overall high level of suppression of Phytophthora damping‐off and root rot of cucumber (66–85% healthy seedlings) and relatively low level of suppression of Rhizoctonia damping‐off of radish (18–38% healthy seedlings). Bacterial treatments also resulted in higher plant fresh masses. Seed coating with irradiated peat formulation of a mixture of three bacteria resulted in superior control of Phytophthora damping‐off and root rot of cucumber and much higher plant fresh masses. The presence of genes for biosynthesis of phenazine‐1‐carboxylic acid, 2,4‐diacetylphloroglucinol, pyrrolnitrin and pyoluteorin was confirmed in Pseudomonas strains, and that of fengycin, bacillomycin, bacilysin, surfactin and iturin A in B. subtilis Bs 8B‐1. All three strains produced HCN, salicylic acid, indole‐3‐acetic acid, protease and β‐1,3‐glucanase. Both Pseudomonas strains produced siderophores and only P. fluorescens Pf 9A‐14 showed phosphate solubilisation and chitinase activity. All three strains inhibited pathogen growth by producing volatiles, and gas chromatography–mass spectrometry analysis revealed eight compounds in Pf 9A‐14, 10 in Bs 8B‐1 and 4 in Psp 8D‐45, some with known antifungal activity. The antagonistic and plant‐growth promotion activities of these strains might be due to production of antibiotics, metabolites, lytic enzymes or phytohormones.     

Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.     

Modelling and refinement of the conformation of mycosubtilin in solution from two-dimensional NMR data

The conformation in solution of mycosubtilin, an antifungal lipopeptide [cyclo(L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser-beta-amino acid)]has been probed by two-dimensional nuclear magnetic resonance and restrained energy minimization. Several structures have been proposed belonging to the same family with minor local variations related to different orientations of amide planes. The molecular topology was found to be completely different from that of iturin A, an analogue which exhibits quite different biological properties. The cyclic peptide of mycosubtilin is shown to be rather rigid in the region of L-proline and stabilized by C7 structures; in contrast, the neighbourhood of D-tyrosine-2 was found to be more flexible. The validity of our models is discussed first in terms of distance violation and second on the basis of reconstructed NOE spectroscopy maps. The different limitations towards higher-resolution structures are discussed.     

Instability of actinomadurae protoplasts generated by lytic enzyme treatment

The effect of lytic enzyme treatment upon protoplast formation and reversion in three species of Actinomadura has been determined. Incubation in the presence of lytic enzyme L2 generates large protoplasts (4 μm diameter) which remain intact for only 2h. In comparison, protoplasts formed by the degradative effects of the enzymes lysozyme and L1 are smaller (2 μm diameter), and remain stable for up to 18h. This results in a greater efficiency of regeneration. Lytic enzyme L2 has been shown to contain impurities, including proteolytic activity, which may affect cell wall regeneration.