MALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens

in Journal of the American Society for Mass Spectrometry (in press)

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial ... [more ▼]

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDIFTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology. [less ▲]

Analysis of calcium-induced effects on the conformation of fengycin

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 ▲]

N-Acetylglucosamine-Mediated Inhibition of Siderophore Biosynthesis Counteracts the suicidal Tendencies of Streptomyces coelicolor

Poster (2012, October)

Distribution and identification of molecular interactions between tomato roots and bacterial biofilms

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 ▲]

Importance of surfactin for plant resistance induction by Bacillus isolates

Conference (2012, June 27)

Calcium-induced conformational changes of the elicitor and membrane-active fengycin

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 ▲]

Design of a biofilm reactor comprising a metal structured packing for the production of lipopeptides by B. subtilis

Diverse speeche and writing (2012)

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 ▲]

Newly discovered natural functions for cyclic lipopeptides from rhizobacteria

Conference (2012)

Bacillus life in the rhizosphere: impact on lipopeptide signature and biocontrol potential

Conference (2012)

Effect of environmental factors on the interaction plant-pathogen-Bacillus amyloliquefaciens S499

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