When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.

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 H 2 O 2 , followed by differential expression of nine defense-related genes and stimulation of chitinase and b-1,3-glucanase activities. Inhibition of OGAinduced 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 b-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.

Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response.

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"Ropiness" is a bacterial alteration in wines, beers, and ciders, caused by ␤-glucan-synthesizing pediococci. A single glucosyltransferase, Gtf, controls ropy polysaccharide synthesis. In this study, we show that the corresponding gtf gene is also present on the chromosomes of several strains of Oenococcus oeni isolated from nonropy wines. gtf is surrounded by mobile elements that may be implicated in its integration into the chromosome of O. oeni. gtf is expressed in all the gtf ؉ strains, and ␤-glucan is detected in the majority of these strains. Part of this ␤-glucan accumulates around the cells forming a capsule, while the other part is liberated into the medium together with heteropolysaccharides. Most of the time, this polymer excretion does not lead to ropiness in a model medium. In addition, we show that wild or recombinant bacterial strains harboring a functional gtf gene (gtf ؉ ) are more resistant to several stresses occurring in wine (alcohol, pH, and SO 2 ) and exhibit increased adhesion capacities compared to their gtf mutant variants."Ropiness" or "oiliness" is one of the major bacterial alterations in wine. It has no impact on human health. However, the viscosity of the spoiled wines prevents their commercialization (43,54,55). Many microbial species have been isolated from ropy wines: Streptococcus mucilaginous and Leuconostoc and Lactobacillus species (15,18,34,36,53,55). However, the species most often incriminated is Pediococcus parvulus (15,31,48,53). Different strains have been isolated from red and white ropy wines from the Bordeaux region in France or from Basque country ropy ciders and were then shown to cause ropiness when grown in model media. These strains were first called Pediococcus cerevisiae (31), later classified as Pediococcus damnosus by DNA/DNA hybridizations (15, 32), and then finally classified as Pediococcus parvulus based on 16S RNA sequencing (53). The increase in viscosity of wine or model media caused by P. parvulus is due to the production of a high-molecular-weight ␤-glucan. This fibrillar polymer consists of a trisaccharide repeating unit with a ␤-1,3-linked glucosyl backbone and branches made up of single ␤-1,2-linked D-glucopyranosyl residues (16,17,28). The level of production of soluble ␤-glucan by pediococci is low (200 mg/liter at most) and is only slightly modified by external growth parameters (49, 50). Polymer production is controlled by a single transmembrane glucosyltransferase, Gtf, a 567-amino-acid, 65-kDa protein that polymerizes glucosyl residues from UDP glucose (53). Gtf exhibits significant homology with Tts (32% identical amino acids), an inverting glucosyltransferase produced by Streptococcus pneumoniae type 37, which synthesizes a branched ␤-1,3 ␤-1,2 glucan whose structure is very close to that of the glucan causing ropiness (1, 29). Actually, the antibodies targeting the type 37 polysaccharide capsule also agglutinate ropy pediococci (51). The gtf gene encoding Gtf is highly conserved (99.9% identity) in wine-and cider-spoiling bacteria, as it...

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