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See detailAnalysis of calcium-induced effects on the conformation of fengycin
Nasir, Mehmet Nail ULg; Laurent, Pascal ULg; Flore, Christelle ULg et al

in Spectrochimica Acta Part A : Molecular and Biomolecular Spectroscopy (2013), 110

A combination of CD, FT-IR, NMR and fluorescence spectroscopic techniques was applied to elucidate the conformation of fengycin, a natural lipopeptide with antifungal and eliciting activities, in a ... [more ▼]

A combination of CD, FT-IR, NMR and fluorescence spectroscopic techniques was applied to elucidate the conformation of fengycin, a natural lipopeptide with antifungal and eliciting activities, in a membrane-mimicking environment and to investigate the effect of calcium ions on the conformation. We mainly observed that fengycin adopts a turn conformation and that the side chain of glutamate residues plays a key role on the stabilization of the peptide ring backbone conformation. More particularly, the binding of calcium ions by the carboxylic moieties has a consequence on the environment of the tyrosine residues. Our data suggest also an arrangement of fengycin molecules into “-sheet like micelles” in a membrane-mimicking environment and the enhancement of this aggregating effect in presence of calcium ions. The modulation of the fengycin conformation by the environmental conditions may influence its biological properties. [less ▲]

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See detailImplementation of structured metal packing for the design of biofilm reactor : analysis by high energy X-ray tomography and application to the production of lipopeptides by Bacillus subtilis
Zune, Quentin ULg; Soyeurt, Delphine; Ongena, Marc ULg et al

Poster (2012, October 08)

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce ... [more ▼]

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce high added value molecules. Thus, it is required to design new bioreactors able to promote the growth of the biomass on the form of a biofilm and to identify the key physico-chemical parameters involved in order to optimize the bioprocess. 2. Aim of this study was to investigate a pilot-scale biofilm reactor comprising a metal structured packing promoting growth of Bacillus subtilis as a biofilm for the production of lipopeptides, high added value compounds with high surface active properties. 3. In this work, the mechanical stirring system of a 20L stirred tank bioreactor has been removed and replaced by a metal structured packing positioned in the headspace of the vessel above a liquid phase. The culture medium is continuously recirculated on the packing thanks to a peristaltic pump and air supply is performed just above the liquid phase under the packing. High energy X-ray tomography was used to estimate non-invasively the biofilm distribution inside the packing and permitted to define parameters that affect scale-up. Performances of the biofilm reactor were compared with a submerged culture in a stirred tank reactor in terms of lipopeptides production. 4. After 72 hours of fermentation, 94 % of the total biomass adheres onto the metal packing on the form of a biofilm. The colonization of this latter has been visualized non-invasively by X-ray tomography directly inside the packing and shows a conical repartition of the biofilm mass (about 25% of the total volume of the packing) as well as the presence of clogging. However, unlike the submerged culture, no foam formation appeared during fermentation and surfactin yield reaches 345,4 ± 32,8 mg / L for the biofilm reactor against 277,3 ± 34,4 mg / L in the stirred tank reactor. 5. In conclusion, this experimental setting leads to a major technological progress avoiding foam formation and increasing surfactin production. Nevertheless, significant improvements are required at the level of the biofilm distribution in thin layers inside the packing in order to increase mass transfer and lipopeptides recoveries. Further investigations will be devoted to the optimization of the physico-chemical parameters involved in biofilm distribution. [less ▲]

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See detailImplementation of structured metal packing for the design of biofilm reactor : analysis by high energy X-ray tomography and application to the production of lipopeptides by Bacillus subtilis
Zune, Quentin; Soyeurt, Delphine; Ongena, Marc ULg et al

Poster (2012, October 08)

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce ... [more ▼]

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce high added value molecules. Thus, it is required to design new bioreactors able to promote the growth of the biomass on the form of a biofilm and to identify the key physico-chemical parameters involved in order to optimize the bioprocess. 2. Aim of this study was to investigate a pilot-scale biofilm reactor comprising a metal structured packing promoting growth of Bacillus subtilis as a biofilm for the production of lipopeptides, high added value compounds with high surface active properties. 3. In this work, the mechanical stirring system of a 20L stirred tank bioreactor has been removed and replaced by a metal structured packing positioned in the headspace of the vessel above a liquid phase. The culture medium is continuously recirculated on the packing thanks to a peristaltic pump and air supply is performed just above the liquid phase under the packing. High energy X-ray tomography was used to estimate non-invasively the biofilm distribution inside the packing and permitted to define parameters that affect scale-up. Performances of the biofilm reactor were compared with a submerged culture in a stirred tank reactor in terms of lipopeptides production. 4. After 72 hours of fermentation, 94 % of the total biomass adheres onto the metal packing on the form of a biofilm. The colonization of this latter has been visualized non-invasively by X-ray tomography directly inside the packing and shows a conical repartition of the biofilm mass (about 25% of the total volume of the packing) as well as the presence of clogging. However, unlike the submerged culture, no foam formation appeared during fermentation and surfactin yield reaches 345,4 ± 32,8 mg / L for the biofilm reactor against 277,3 ± 34,4 mg / L in the stirred tank reactor. 5. In conclusion, this experimental setting leads to a major technological progress avoiding foam formation and increasing surfactin production. Nevertheless, significant improvements are required at the level of the biofilm distribution in thin layers inside the packing in order to increase mass transfer and lipopeptides recoveries. Further investigations will be devoted to the optimization of the physico-chemical parameters involved in biofilm distribution. [less ▲]

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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Ongena, Marc ULg et al

Conference (2012, September 05)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

Detailed reference viewed: 65 (12 ULg)
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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Smargiasso, Nicolas ULg et al

Conference (2012, September)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

Detailed reference viewed: 54 (10 ULg)
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See detailImportance of surfactin for plant resistance induction by Bacillus isolates
Cawoy, Hélène ULg; Mariutto, Martin; Jourdan, Emmanuel et al

Conference (2012, June 27)

Detailed reference viewed: 41 (15 ULg)
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See detailCalcium-induced conformational changes of the elicitor and membrane-active fengycin
Nasir, Mehmet Nail ULg; Lins, Laurence ULg; Ongena, Marc ULg et al

Poster (2012, April)

Fengycin is a natural lipopeptide synthetized by Bacillus subtilis strains. It is characterized by strong antifungal and low hemolytic activities. It seems also play a role in the promoting of elicitor ... [more ▼]

Fengycin is a natural lipopeptide synthetized by Bacillus subtilis strains. It is characterized by strong antifungal and low hemolytic activities. It seems also play a role in the promoting of elicitor activities of other compounds. The target of the biological activities of fengycin is supposed to be plasma membrane of sensitive cells. Even though the natural fengycin from has been discovered 25 years ago, nowadays, there is an increase of interest for this compound because of its potent applications. Until 15 years ago, the primary structure of fengycin was a matter of open debate before the publication of the corrected structure obtained by nuclear magnetic resonance and mass spectroscopy techniques. Although the infrared and ultraviolet absorption spectra of the lipopeptide were measured, no detailed analysis of these data was performed probably because of the unconventional sequence of the lipopeptide making these kinds of analyses complicated. In this work, our attempt was to analyze the conformational properties of fengycin as well as the calcium-induced changes using two complementary spectroscopic methods, Fourrier transformed infrared spectroscopy (FTIR) and circular dichroism (CD). In a first step, we have characterized the conformational properties of pure fengycin. The lipopeptide adopts turn conformation in trifluoroethanol, a membrane-mimicking solvent. D-aminoacids seem to be involved in intra molecular hydrogen bonds. In a second step, we have investigated the role played by Ca2+ ions on the possible conformational changes of fengycin. The addition of calcium gives rise to important modifications of the conformation. As fengycin has two glutamate residues, calcium is supposed to bind to their side chains. In conclusion, we have demonstrated that the conformation of fengycin is closely depending of the environment and the presence of calcium ions play an important role on the conformational changes of the lipopeptide. Moreover, spectra obtained both FTIR and CD methods ascertain the presence of turn conformation. [less ▲]

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See detailHow does elicitor and antimicrobial fengycin interact with plasma membranes of sensitive cells ?
Nasir, Mehmet Nail ULg; Eeman, Marc; Lins, Laurence ULg et al

Conference (2012, April)

Fengycin is characterized by its strong antifungal and low hemolytic activities. It has also been recently demonstrated that it has plant elicitor properties and is also able to enhance the elicitors’ ... [more ▼]

Fengycin is characterized by its strong antifungal and low hemolytic activities. It has also been recently demonstrated that it has plant elicitor properties and is also able to enhance the elicitors’ activity of surfactin. The cell target of its biological activities is supposed to be plasma membrane. In spite of these interesting biological activities, fengycin has not been extensively investigated probably because of the difficulties related to its production. In a first time; we have characterized the interfacial properties of fengycin by tensiometry measurements and demonstrated that this surface activity was pH-dependent. In a second time; we have investigated the interactions of the lipopeptide with membrane lipids using model membranes such as Langmuir monolayers and multilammelar vesicles (MLVs). Our results indicate that the lipopeptide was able to penetrate into different lipid monolayers showing a preference for sterol-containing monolayers. In order to better understand the mechanism of the interactions of fengycin with membranes at the molecular level, MLVs with or without fengycin have been analyzed by spectroscopic techniques. We have shown that conformational changes of the lipopeptide occurred in the presence of lipids and they were more significant in the presence of sterol. Moreover, tyrosine residues of the lipopeptide seem to play an important role in these interactions. In conclusion, we have determined that the surface-active behavior as well as the conformation of fengycin depends on its environment. We have also showed that the lipopeptide does not interact with all class of lipids in the same way and presents a preference for sterols. The presence of key groups within peptide cycle has also been supposed for the biological activities of the lipopeptide. [less ▲]

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See detailDesign of a biofilm reactor comprising a metal structured packing for the production of lipopeptides by B. subtilis
Zune, Quentin ULg; Ongena, Marc ULg; Toye, Dominique ULg et al

Poster (2012, February 08)

Abstract : The design of a new single species biofilm bioreactor has been investigated. Bacillus subtilis S499 has been chosen as a model organism for the production of lipopetides. Nevertheless ... [more ▼]

Abstract : The design of a new single species biofilm bioreactor has been investigated. Bacillus subtilis S499 has been chosen as a model organism for the production of lipopetides. Nevertheless, considering the surface active properties for this kind of metabolite, processes based on submerged culture in stirred-tank bioreactor involve the use of important amount of antifoam and therefore downstream processes are tedious. In this work, an original process was developed with an experimental setting leading to the suppression of foam formation during the culture. B. subtilis S499 makes a biofilm on a stainless steel structured packing in the top of a bioreactor, nutrient and oxygen supply being carried out by the media recirculation as liquid film on the packing. Lipopeptides secreted by biofilm are accumulated in the liquid phase under the packing and can reach concentrations as high as 800 mg/l. The colonization of the packing by the biofilm has been monitored by X-ray tomography. [less ▲]

Detailed reference viewed: 111 (27 ULg)
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See detailBacillus subtilis: biocontrol and growth promotion
Ongena, Marc ULg

Conference (2012)

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See detailUnsuspected Control of Siderophore production by N-acetylglucosamine in Streptomycetes
Craig, Matthias ULg; Lambert, Stéphany ULg; Jourdan, Samuel ULg et al

in Environmental Microbiology Reports (2012)

Detailed reference viewed: 54 (25 ULg)
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See detailNewly discovered natural functions for cyclic lipopeptides from rhizobacteria
Ongena, Marc ULg; Henry, G.; Deleu, Magali ULg et al

Conference (2012)

Detailed reference viewed: 20 (1 ULg)
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See detailEffect of environmental factors on the interaction plant-pathogen-Bacillus amyloliquefaciens S499
Pertot, I.; Hosni, T.; Pedrotti, L. et al

in Bulletin OILB/SROP = IOBC/WPRS Bulletin (2012), sous presse

Detailed reference viewed: 39 (3 ULg)