Structure of an exocellular β-d-glucan from Pediococcus sp., a wine lactic bacteria

"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...

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Characterization ofgtf, a Glucosyltransferase Gene in the Genomes ofPediococcus parvulusandOenococcus oeni, Two Bacterial Species Commonly Found in Wine

Dols‐Lafargue

Lee

Marrec

et al. 2008

“…EPS-produc-ing LAB are responsible for a "ropy" phenotype characterized by a viscous and thick texture observed in spoiled alcoholic beverages, such as beer, cider, and wine (20,39,68). Despite this, the production of EPS has many beneficial effects both in the food industry and on individuals' health.…”

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confidence: 99%

Association of Beta-Glucan Endogenous Production with Increased Stress Tolerance of Intestinal Lactobacilli

Stack

Kearney

Stanton

et al. 2010

122

The exopolysaccharide beta-glucan has been reported to be associated with many health-promoting and prebiotic properties. The membrane-associated glycosyltransferase enzyme (encoded by the gtf gene), responsible for microbial beta-glucan production, catalyzes the conversion of sugar nucleotides into beta-glucan. In this study, the gtf gene from Pediococcus parvulus 2.6 was heterologously expressed in Lactobacillus paracasei NFBC 338. When grown in the presence of glucose (7%, wt/vol), the recombinant strain (pNZ44-GTF ؉ ) displayed a "ropy" phenotype, while scanning electron microscopy (SEM) revealed strands of polysaccharidelinking neighboring cells. Beta-glucan biosynthesis was confirmed by agglutination tests carried out with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. Further analysis showed a ϳ2-fold increase in viscosity in broth media for the beta-glucan-producing strain over 24 h compared to the control strain, which did not show any significant increase in viscosity. In addition, we analyzed the ability of beta-glucan-producing Lactobacillus paracasei NFBC 338 to survive both technological and gastrointestinal stresses. Heat stress assays revealed that production of the polysaccharide was associated with significantly increased protection during heat stress (60-fold), acid stress (20-fold), and simulated gastric juice stress (15-fold). Bile stress assays revealed a more modest but significant 5.5-fold increase in survival for the beta-glucan-producing strain compared to that of the control strain. These results suggest that production of a beta-glucan exopolysaccharide by strains destined for use as probiotics may afford them greater performance/protection during cultivation, processing, and ingestion. As such, expression of the gtf gene may prove to be a straightforward approach to improve strains that might otherwise prove sensitive in such applications.

“…1 H and 13 C nuclear magnetic resonance spectroscopy revealed a glucan with a ␤-1,3 backbone and additional ␤-1,2-linked glucose at every second glucose molecule of the ␤-1,3-glucan as described previously (20,38,58). 1 H and 13 C NMR chemical shifts (␦, ppm) and coupling constants (J, Hz) for the EPS of Pediococcus parvulus strain B399 are summarized in Table 4.…”

Section: Resultsmentioning

confidence: 57%

“…Whereas the ␤-glucan of the exopolysaccharide possesses a ␤-1,3-linked glucosyl backbone with branches made up of single ␤-1,2-linked D-glucopyranosyl residues (38), the cell wall of yeasts mainly contains ␤-1,3 and ␤-1,6 but also variably linked ␤-glucans, too (33). Thus, by means of suitable enzymes not only could the slime of Pediococcus parvulus be degraded but also the undesirable growth of yeasts could be inhibited.…”

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confidence: 99%

β-1,3-Glucanase from Delftia tsuruhatensis Strain MV01 and Its Potential Application in Vinification

Blättel¹,

Larisika²,

Pfeiffer³

et al. 2011

During vinification microbial activities can spoil wine quality. As the wine-related lactic acid bacterium Pediococcus parvulus is able to produce slimes consisting of a ␤-1,3-glucan, must and wine filtration can be difficult or impossible. In addition, the metabolic activities of several wild-type yeasts can also negatively affect wine quality. Therefore, there is a need for measures to degrade the exopolysaccharide from Pediococcus parvulus and to inhibit the growth of certain yeasts. We examined an extracellular ␤-1,3-glucanase from Delftia tsuruhatensis strain MV01 with regard to its ability to hydrolyze both polymers, the ␤-1,3-glucan from Pediococcus and that from yeast cell walls. The 29-kDa glycolytic enzyme was purified to homogeneity. It exhibited an optimal activity at 50°C and pH 4.0. The sequencing of the N terminus revealed significant similarities to ␤-1,3-glucanases from different bacteria. In addition, the investigations indicated that this hydrolytic enzyme is still active under wine-relevant parameters such as elevated ethanol, sulfite, and phenol concentrations as well as at low pH values. Therefore, the characterized enzyme seems to be a useful tool to prevent slime production and undesirable yeast growth during vinification.