Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air-water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air-water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.     

Mycosubtilin and surfactin are efficient,low ecotoxicity molecules for the biocontrol of lettuce downy mildew

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L^?1 in Microtox assays and 6 mg L^?1 in Daphnia magna immobilization tests for mycosubtilin and 125 mg L^?1 and 25 mg L^?1 for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L^?1 of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50 mg L^?1) gave the same result on B. lactucae development as 100 mg L^?1 of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.     

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 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.     

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.     

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.     

Setting up and modelling of overflowing fed-batch cultures of Bacillus subtilis for the production and continuous removal of lipopeptides

This work is related to the set-up of overflowing exponential fed-batch cultures (O-EFBC) derived from carbon limited EFBC dedicated to the production of mycosubtilin, an antifungal lipopeptide belonging to the iturin family. O-EFBC permits the continuous removal of the product from the bioreactor achieving a complete extraction of mycosubtilin. This paper also provides a dynamical Monod-based growth model of this process that is accurate enough to simulate the evolution of the specific growth rate and to correlate it to the mycosubtilin specific productivity. Two particular and dependant phenomena related to the foam overflow are taken into account by the model: the outgoing flow rate of a broth volume and the loss of biomass. Interestingly, the biomass concentration in the foam was found to be lower than the biomass concentration in the bioreactor relating this process to a recycling one. Parameters of this model are the growth yield on substrate and the maximal specific growth rate estimated from experiments led at feed rates of 0.062, 0.071 and 0.086h(-1). The model was extrapolated to five additional experiments carried out at feed rates of 0.008, 0.022, 0.040, 0.042 and 0.062h(-1) enabling the correlation of the mean specific growth rates with productivity results. Finally, a feed rate of 0.086h(-1) corresponding to a mean specific growth rate of 0.070h(-1) allowed a specific productivity of 1.27mg of mycosubtiling(-1) of dried biomassh(-1).     

Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities

A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.     

Mycosubtilin Overproduction by Bacillus subtilis BBG100 Enhances the Organism's Antagonistic and Biocontrol Activities

A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.     

Mycosubtilins B and C: minor antibiotics from mycosubtilin-producer Bacillus subtilis

Mycosubtilins B and C were isolated from the culture medium of Bacillus subtilis. The acid hydrolysates of these new antifungal antibiotics, like mycosubtilin, contain alpha-amino acids (Asp3, Glu1, Pro1, Ser1 and Tyr1) and a mixture of iso-C16, n-C16, iso-C17 and anteiso-C17 beta-amino acids. Mycosubtilins B and C differ by the presence of a carboxyl group and of a carboxymethyl group, respectively, instead of a carboxamide group in previously described mycosubtilin.     

大豆根腐病生防菌KJB04-11的鉴定及其产生的脂肽类抗生素

从大豆根围筛选到1株对尖孢镰刀菌和立枯丝核菌都具有很好拮抗作用的菌株KJB04-11,经形态观察、生理生化特征和16SrDNA序列分析,属于枯草芽孢杆菌(Bacillussubtilis)。具有抗菌活性的KJB04-11发酵液无菌滤液对热和酸碱具有较强的稳定性。采用SephadexG-25柱层析、反相HPLC和冷冻干燥从KJB04-11发酵液中分离纯化了抗菌活性成分。由红外光谱、MALDI-TOF-MS、氨基酸组成及脂肽合成酶基因扩增结果推测该菌株产生的抗菌物质为C16、C17的mycosubtilin和C15的surfactin。田间试验表明,大豆种子经KJB04-11发酵液包衣处理对大豆根腐病防效为53.6%,大豆产量提高12.5%。     

The cell biology of cell-in-cell structures

For decades, authors have described unusual cell structures, referred to as cell-in-cell structures, in which whole cells are found in the cytoplasm of other cells. One well-characterized process that results in the transient appearance of such structures is the engulfment of apoptotic cells by phagocytosis. However, many other types of cell-in-cell structure have been described that involve viable non-apoptotic cells. Some of these structures seem to form by the invasion of one cell into another, rather than by engulfment. The mechanisms of cell-in-cell formation and the possible physiological roles of these processes will be discussed.     

Enigmatic ichnites in the middle triassic of Southern Spain

In southern Spain, within the Chiclana de Segura Formation of red detrital fades, enigmatic structures were found whose origins are discussed here. Each structure is composed of the union of elongated traces, two laterals and a shorter central one. All are concave epireliefs at the top of a sandstone bed whose age is assumed to be Ladinian (Triassic). The sediments formed within a fluvial environment. We describe these imprints, their structures and measurements, and discuss their orientations and associations. The traces probably have an organic origin, and are most likely tracks made by a vertebrate, possibly an archosaur, although no osseous remains have been found to confirm this interpretation.     

A Review of Nature-Inspired Algorithms

The study of bionics bridges the functions, biological structures and organizational principles found in nature with our modern technologies, and numerous mathematical and metaheuristic algorithms have been developed along with the knowledge transferring process from the lifeforms to the human technologies. Output of bionics study includes not only physical products, but also various computation methods that can be applied in different areas. People have learnt from biological systems and structures to design and develop a number of different kinds of optimisation algorithms that have been widely used in both theoretical study and practical applications. In this paper, a number of selected nature-inspired algorithms are systematically reviewed and analyzed. Though the paper is mainly focused on the original principle behind each of the algorithm, their applications are also discussed.     

雄蚤生殖节的扫描电镜观察

本文利用扫描电镜对雄蚤生殖节表面细微结构进行了观察,发现了一些光镜下不易看到的纹理状结构和小棘及一些感器.这些微小的纹理状结构和小棘在不同蚤种之间有差异,这种差异具有相对的稳定性,可望用于蚤的亚显微水平的分类.通过对感器的观察,发现生殖节上具有大量的毛形感器,另外还观察到有锥形感器、短锥形感器、钟形感器、栓锥感器、腔锥感器及刺形感器等.这些感器的数量分布及有无因种而异.这些感器的存在与交配行为密切相关,作者对此也进行了讨论.     

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l^?1. On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l^?1). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.     

On the identification of feather structures in stem-line representatives of birds: evidence from fossils and actuopalaeontology

Dinosaurs with fossilized filamentous integument structures are usually preserved in a highly flattened state. Several different feather types have been described on this basis, but the two-dimensional preservation of specimens during fossilization makes the identification of single feather structures difficult due to overlapping feather structures in vivo. Morphological comparison with the diversity of recent feather types is therefore absolutely vital to avoid misinterpretation. To simulate the preservation process, a cadaver of recent Carduelis spinus (European siskin) was flattened in a printing press. Afterwards, the structure of the plumage was compared with the morphology of a single body feather from the same specimen. In comparison with the single feather, the body plumage of the flattened bird looked rather filamentous. It was almost impossible to identify single structures, and in their place, various artefacts were produced. The investigation of plumage in a specimen of the Mesozoic bird Confuciusornis sanctus reveals similar structures. This indicates that flattening of specimens during fossilization amplifies the effect of overlapping among feathers and also causes a loss of morphological detail which can lead to misinterpretations. The results are discussed in connection with some dubious feather morphologies in recently described theropods and basal birds. Based on recent feather morphology, the structure of so-called proximal ribbon-like pennaceous feathers (PRPFs) found in many basal birds is reinterpreted. Furthermore, the morphology of a very similar-looking feather type found in the forelimb and tail of an early juvenile oviraptorosaur is discussed and diagnosed as the first feather generation growing out of the feather sheath. Thus, the whole plumage of this theropod might represent neoptile plumage.     

NMR structure of active and inactive forms of the sterol-dependent antifungal antibiotic bacillomycin L.

The antifungal antibiotic lipopeptide bacillomycin L [cyclo-(L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6++ +-L-Thr7-beta-amino fatty acid)] from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol-phospholipid dependence on biomembranes. This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (L-Asp1, D-Tyr2) di-O-methylated form. The structures were calculated under NMR-derived restraints using molecular-dynamic simulated-annealing protocols starting from a random array of atoms. The structure of the native antibiotic is spread over different conformers in which two families are recognized. It was found that most structures have dihedral phi and psi angles defining a type-II' beta-turn including amino acids 5-8, in certain cases stabilized by a 8HN-5CO hydrogen bond, whereas a minority of structures adopt an inverse gamma-turn including amino acids 6-8, stabilized in all cases by an 8HN-6CO hydrogen bond. The di-O-methylation of L-Asp1 and D-Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures. The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved D-Tyr2 position.