Alain Heyraud

Oligogalacturonide signal transduction, induction of defense-related responses and protection of grapevine against Botrytis cinerea

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H2O2, followed by differential expression of nine defense-related genes and stimulation of chitinase and β-1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and β-1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.

GRAM-SCALE SYNTHESIS OF RECOMBINANT CHITOOLIGOSACCHARIDES IN ESCHERICHIA COLI

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.

Extraction of highly gelling pectic substances from sugar beet pulp and potato pulp : influence of extrinsic parameters on their gelling properties

Abstract Sugar beet and potato pulps were considered as potential new sources of pectins because of their high pectin content, from 15 to 30% on a dry matter basis, and their availability in large quantities. Up to now, no pectins with gel forming properties comparable to the pectins extracted from apple pomace and citrus peels have ever been extracted from these by-products. In the present work, an extraction process for each by-product has been developed which maintains the structural integrity of the pectins as much as possible. These procedures yielded products which possessed both a high pectic substance content with a low degree of esterification and a high gelling ability in the presence of calcium. The composition of the extracted products was determined and the effect of extrinsic factors such as concentration of the extracted product, calcium content, sequestrant content and hydration temperature, on gel properties was evaluated by rheological measurements.

Stereochemical course of the hydrolysis reaction catalyzed by chitinases Al and D from Bacillus circulans WL‐12

Chitinases A1 and D were purified from the periplasmic proteins produced by Escherichia coli HB101 harbouring recombinant plasmids carrying respectively the chiA and chiD genes of Bacillus circulans WL‐12. HPLC analysis indicated that during the hydrolysis of chitotriose, both chitinases initially produce N‐acetylglucosamine and only one anomer of chitobiose. 1H NMR spectroscopy of the hydrolysis of chitotetraitol showed that this anomer corresponds to β‐chitobiose, demonstrating that chitinases Al and D act by a molecular mechanism that retains the anomeric configuration. This mechanism is similar to that of lysozymes although both chitinases belong to a family of proteins sharing no demonstrable amino acid sequence similarity with lysozymes.

Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-β-d-arabino-hexuronic acid

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.

Structural characterization and rheological properties of an extracellular glucuronan produced by a Rhizobium meliloti M5N1 mutant strain

The mutant strain M5N1 C.S. (NCIMB 40472) of Rhizobium meliloti M5N1 is able to produce during fermentation a partially acetylated extracellular (1-->4)-beta-D-glucuronan. At low concentration (1 g.l-1), in the presence of monovalent cations, this new glucuronate behaves as a thickening agent, whereas at higher concentration a thermoreversible gel is obtained. With such divalent cations as Ca2+, a thermally stable gel can be formed.

Production of lewis x tetrasaccharides by metabolically engineered Escherichia coli

Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram‐scale by high‐cell‐density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for α‐3 fucosyltransferase and the Neisseria meningitidis lgtB gene for β‐4 galactosyltransferase. The first compound Galβ‐4(Fucα‐3)GlcNAcβ‐4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin‐synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Galβ‐4(Fucα‐3)GlcNAcβ‐3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for β‐3 N‐acetylglucosaminyltransferase.

Galactomannans from Brazilian seeds: characterization of the oligosaccharides produced by mild acid hydrolysis

Galactomannans with Man:Gal ratios ranging from 1.1:1 to 3:1, obtained from the seeds of Mimosa scabrella, Stryphnodendron barbatiman, Schizolobium parahybum and Schizolobium amazonicum, were submitted to mild acid hydrolysis. The products were fractionated by gel permeation chromatography on BioGel P2 yielding fractions with degrees of polymerization (DP) of 1 to 6. Those with DP 2 to 6 from each species were analysed by ion-exchange high-performance liquid chromatography and characterized by 13C- and 1H-nuclear magnetic resonance (NMR) spectroscopy. The distribution of the oligosaccharides of each degree of polymerization was very similar for the products from S. parahybum and S. amazonicum, indicating the same D-galactosyl distribution on the D-mannan backbone, in agreement with the 13C-NMR splitting in the C4 region of the D-mannosyl units in the original polymers. The hydrolytic conditions adopted allowed characterization of compounds that are not generally produced by enzymatic treatments. The results show that the structures of the oligosaccharides, even if there is a preferential hydrolysis of Gal-Man linkages, reflect the composition of the parent polymer.

Characterization of exopolysaccharides produced by rhizobacteria

SummaryBacteria isolated from the rhizosphere, the rhizobacteria, of sorghum, pearl millet, wheat, alfalfa and rice were screened for the production of exopolysaccharide (EPS). Nearly a quarter of the strains produced exopolysaccharides, either capsular or hydrosoluble slime. A majority of the isolates produced slime. Physico-chemical analyses have indicated the ability of certain diazotrophic Pseudomonas paucimobilis isolates from millets and sorghum to produce unique types of EPS, which are highly viscous and thermostable.

A (1→4)-β-D-glucuronan excreted by a mutant of the Rhizobium meliloti M5N1 strain

Abstract A mutant of the R. meliloti M5N1 strain has been selected. This strain, R. meliloti M5N1 CS (NCIMB 40472), excretes an extracellular material composed of 2-O-Ac-β-GlcpA, 3-O-Ac-β-GlcpA, 2,3-di-O-Ac-β-GlcpA and three species of β-GlcpA residues 1→4 linked. For the culture conditions used, the weight average molecular weight of the polymer varied in the range of 6 × 104 < Mw < 4 × 105. High molecular weight glucuronate forms thermoreversible gels at 5 g L−l. In the presence of divalent cation such as Ca2+ or trivalent cations such as Cr3+ or Fe3 +, cross linking of the polymer occurs. This polysaccharide is the first exocellular (1→4)-β-D-glucuronan produced by a R. meliloti strain.