Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize

Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification

Evaluation of a cereal milling by-product for the low cost production of Bacillus thuringiensis kurstaki in submerged fermentation

With the idea of finding a cheap medium for the mass production of Bacillus thuringiensis (Bt), a cereal milling by product (CMB) was evaluated and was shown efficient to be used as a source of carbohydrates, proteins and minerals for the production of δ-endotoxins in submerged fermentation of a new strain of Bacillus thuringiensis kurstaki “Lip”. The results obtained in 1000 mL shake flasks experiments showed that the toxin proteins concentration produced in the 6% (w/v) CMB medium was 2.4 fold and 1.54 fold greater than the values reached in the Anderson medium and in a standard semi synthetic medium respectively, while there was no significant difference between the cell, spore and crystal counts or between the protein profiles of the “Lip” spore/crystal complex obtained in the three mediums. Considering all three factors, relative yield, productivity and cost, the use of the CMB mono-component medium was proved much more economical for an industrial production of “Lip” than the references mediums. Moreover, the suitability of the medium for large scale production of Bt based bio insecticide was evidenced in a 5 L lab bioreactor

A simple method for the separation of Bacillus thuringiensis spores and crystals

A simple new method, for separating Bacillus thuringiensis crystals from spores and cell debris, is described. The developed purification method uses hexane and low speed centrifugation and does not require any expensive material or reagents

Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization

9siIn host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.openopenSegond D; Abi Khalil E; Buisson C; Daou N; Kallassy M; Lereclus D; Arosio P; Bou-Abdallah F; Nielsen Le Roux C.Segond, D; Abi Khalil, E; Buisson, C; Daou, N; Kallassy, M; Lereclus, D; Arosio, Paolo; Bou Abdallah, F; Nielsen Le Roux, C

IlsA, A Unique Surface Protein of Bacillus cereus Required for Iron Acquisition from Heme, Hemoglobin and Ferritin

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts

Identification de nouveaux facteurs hôtes-dépendants chez Bacillus cereus (caractérisation moléculaire et fonctionnelle d'IlsA, une protéine de surface essentielle pour l'acquisition du fer au cours de l'infection)

PARIS-AgroParisTech Centre Paris (751052302) / SudocSudocFranceF

Diversity of Bacillus cereus sensu lato mobilome

Bacillus cereus sensu lato s.l.) is a group of bacteria displaying close phylogenetic relationships but a high ecological diversity. The three most studied species are Bacillus anthracis, Bacillus cereus sensu stricto and Bacillus thuringiensis. While some species are pathogenic to mammals or associated with food poisoning, Bacillus thuringiensis is a well-known entomopathogenic bacterium used as biopesticide worldwide. B. cereus s.l. also contains a large variety of mobile genetic elements (MGEs)

IS 982 and kin: new insights into an old IS family.

Insertion sequences (IS) are ubiquitous transposable elements with a very simple organization: two inverted repeats flanking a transposase coding gene. IS is one of 26 insertion sequence families known so far. With 70 registered members in the ISFinder database, this family remains somewhat unexplored, despite the association of many of its members with important features such as antibiotic resistance. IS has a fairly simple organization with a mean length of ca. 1 Kb, two inverted repeats with conserved 5' AC 3' ends flanking a transposase coding gene and direct repeats of variable lengths. Its transposase has a RNAse-H like chemistry with an atypical DDE motif. In this study, we first highlight the current knowledge on the IS family by dissecting its registered members and their characteristics. Secondly, we bring new insights into this old, yet uncharted IS family, by exploring its registered elements, as well as the genomic and proteomic databases of bacterial and archaeal strains. This probing showed that the presence and distribution of this family goes far beyond the clear-cut registry of ISFinder database

Analyse moléculaire des gènes cry1A d'une souche de Bacillus thuringiensis et étude de l'interaction des toxines correspondantes dans une modèle de membrane biomimétique

Bacillus thuringiensis (Bt) est une bactérie produisant des inclusions protéiques cristallines à pouvoir insecticide et elle est largement exploitée à l'échelle industrielle. Dans cette étude, des souches de Bt ont été isolées du sol libanais. Nous avons étudié en premier la présence des principaux gènes cry1A codant pour des -endotoxines actives sur les lépidoptères. Les souches possédant ces gènes ont été testées pour leur toxicité sur des larves d'Ephestia kuehniella (E. kuehniella). Une souche nommée Lip, étant quatre fois plus toxique sur ces larves que la référence mondiale Bt subsp. kurstaki HD1, fut sélectionnée pour une étude plus approfondie. Après clonage et séquençage, nous avons identifié une nouvelle toxine de type Cry1Aa : Cry1Aa22 et une nouvelle variante de la toxine Cry1Ac. Ces dernières se sont montrées plus toxiques sur des larves d'E. kuehniella, et plus stables en présence des protéases intestinales de ces larves que Cry1Aa et Cry1Ac de HD1 permettant d'expliquer la toxicité élevée de la souche sauvage. D'autre part, nous avons optimisé la construction d'un modèle de membrane biomimétique incluant la membrane de la bordure en brosse intestinale (BBM) des larves d'E. kuehniella. Ces membranes nous ont servi à l'étude de l'interaction des toxines Cry1Aa et Cry1Ac de Lip et celles de HD1. Les toxines de Lip ont interagit différemment et avec une plus grande affinité avec ces modèles que celles de HD1.Tous ces résultats montrent que Lip est une souche intéressante pour une exploitation industrielle et que le modèle de membrane biomimétique est une alternative permettant la prédiction de l'affinité des toxines Cry.Bacillus thuringiensis (Bt) is a bacterium that synthesizes insecticidal proteic crystallin inclusions and is widely used at an industrial scale. In this study, Bt strains were isolated from Lebanese soil. We studied the presence of the main cry1A genes encoding for -endotoxins active on Lepidoptera. Strains harboring these genes were tested for their toxicity against Ephestia kuehniella (E. kuehniella) larvae. The strain named Lip, being four folds more toxic to the larvae than the reference strain Bt subsp. kurstaki HD1, was selected for further study. We identified a novel Cry1Aa toxin, Cry1Aa22, and a variety of the Cry1Ac toxin after cloning and sequencing of the corresponding genes. These toxins were more toxic to E. kuehniella larvae and more stable in the presence of these larvae's intestinal midgut juice than Cry1Aa and Cry1Ac of HD1. Moreover, we optimized the construction of a biomimetic membrane model based on the intestinal brush border membrane (BBM) of E. kuehniella larvae. These models were used to study the interaction of Cry1Aa and Cry1Ac of Lip and HD1. Toxins of Lip interacted differently and with a greater affinity with these model membranes than toxins of HD1.These results show that Lip is an interesting Bt strain that could be exploited at an industrial scale. On another hand, the biomimetic membrane constructed in this study could be an alternative allowing the prediction of the Cry toxin's affinity.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF