A putative glucan synthase gene dps detected in exopolysaccharide-producing Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider

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Abstract

Some lactic acid bacteria can induce viscosity in wine, beer and cider by production of exopolysaccharides (EPS). A polymerase chain reaction (PCR) assay was previously described for the detection of ropy Pediococcus damnosus strains in wine [J. Appl. Microbiol. 90 (2001) 535]. The primers used in that study, PF5 and PF6, are investigated in addition to new primers which broaden the range of spoiling agents detectable by PCR. Primers PF1 and PF8 allow the amplification of DNA from ropy P. damnosus strains isolated from wine, as was observed with PF5 and PF6. In addition, PF1 and PF8, unlike PF5 and PF6, are able to generate an amplicon using template DNA from a ropy P. damnosus strain isolated from cider and a ropy Oenococcus oeni strain isolated from champagne. Two different ropy Lactobacillus species were also isolated, but their DNA was not amplified using primers PF1 and PF8.

The new primers PF1 and PF8 were chosen from the sequence of gene dps, a putative glucan synthase gene, found across all the ropy P. damnosus strains isolated, from both wine or cider, and also in a ropy O. oeni strain. To our knowledge, this is the first time that an EPS-producing O. oeni strain is described. Glucan biosynthesis was assessed by agglutination tests done with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. The results show that there is a direct correlation between glucan production and detection of gene dps. Therefore, Dps is considered a candidate for the glucan synthase enzyme responsible for EPS production by ropy strains of P. damnosus and O. oeni.

Introduction

Although lactic acid bacteria are beneficial to wine quality since they perform malolactic fermentation, they are regularly responsible for the spoilage of wine and other fermented beverages. One endemic alteration is “ropiness.” This is characterized by a viscous and thick texture of the beverage. The taste of the product remains unchanged but the oily feel degrades the beverage. The spoilage takes place during vinification or during storage and reduces the value of the product. This was first described by Pasteur (1866) and linked to the presence of microorganisms in wine. Slime production also occurs in beer (Van Oevelen and Verachtert, 1979) and cider (Fernández et al., 1995). Since that time, further studies have shown that the lactic acid bacteria Pediococcus damnosus is involved in a vast majority of cases (Lonvaud-Funel and Joyeux, 1988). The viscosity is due to the production of glucan as an exopolysaccharide (EPS) from the residual sugars in wine and cider. (Llaubères et al., 1990, Dueñas-Chasco et al., 1997). To date, little information is known on the genetics of glucan biosynthesis by pediococci.

Studies have focused on the genetic determinants of glucan production by P. damnosus which are located on a plasmid (Lonvaud-Funel et al., 1993). Successive transfers of ropy clones in nonalcoholic medium induces the loss of the ropy phenotype along with the disappearance of a particular plasmid, pF8801 (Lonvaud-Funel et al., 1993). Plasmid pF8801, present in all the ropy strains isolated from wine, was sequenced for the strain P. damnosus IOEB8801 and found to be 5.5 kb long. The sequence revealed genes for the maintenance and transfer of the plasmid (rep and mob) and a putative glucosyltransferase gene named dps (Walling et al., 2001). Based on strong homologies of the deduced protein sequence with known enzymes, the function of the gene product Dps is thought to be the polymerization of glucose residues into glucan (Walling et al., 2001).

The sequence of Dps best aligns with Tts a polysaccharide synthase of Streptococcus pneumoniae type 37 (Llull et al., 1999). It was demonstrated that Tts is responsible for the production of a glucan capsular polysaccharide. The capsule is structurally related to the glucan produced by P. damnosus composed of a linear backbone of 3-β-DGlcp-1 repeating units with monosaccharide side chains of a β-d-Glc-1 linked to C2 of each glucose residue (sophorosyl units). More recently, heterologous expression of the tts gene in several Gram-positive hosts directed the biosynthesis of a glucan capsule. The cells harboring a glucan capsule were agglutinated by S. pneumoniae type 37-specific antibodies (Llull et al., 2001). Based on the homologies between Tts and Dps, it is likely that the plasmid-carrying gene dps is directly involved in the production of glucan by P. damnosus in wine.

The sequence of the plasmid was used to elaborate molecular tools for the detection of ropy P. damnosus strains (Gindreau et al., 2001). A method to purify the DNA from wine microorganisms without any culture step was described and allows the detection of ropy P. damnosus cells by polymerase chain reaction (PCR) with primers PF5 and PF6 (Gindreau et al., 2001). The amplified fragment includes an intergenic region of the plasmid and the beginning of the mob gene. The PCR assay allows the discrimination of P. damnosus ropy strains from non-ropy strains isolated from wine. Primers PF5 and PF6 were previously elaborated and validated using 15 ropy P. damnosus strains, 15 non-ropy P. damnosus strains, and 18 other lactic acid bacteria commonly found in wine (Gindreau et al., 2001).

However, we suspect more species of lactic acid bacteria to produce exopolysaccharides in fermented beverages. The aim of this study was to isolate novel strains from ropy wines and ciders and present new tools for their detection. The limits of the primer set (PF5 and PF6) described in Gindreau et al. (2001) targeting the region ahead of the mob gene on pF8801 were investigated. This study led to the elaboration of a second primer set based on a putative glucan synthase gene, with a broader range of targets. Primers PF1 and PF8 detect not only ropy P. damnosus strains isolated from wine, but also new ropy strains of various species which do not respond to primers PF5 and PF6. Finally, this is the first description of a cross-species putative glucan synthase gene, carried by ropy P. damnosus strains and one Oenococcus oeni strain.

Section snippets

Bacterial strains and growth conditions

Reference strains from the American Type Culture Collection (ATCC) and strains from the collection of the Enology Institute of Bordeaux (IOEB) were used for the development of probes (Lonvaud-Funel et al., 1991) and as positive or negative controls for the PCR amplifications. The IOEB collection of 15 ropy P. damnosus strains and 15 non-ropy P. damnosus strains was used to evaluate the specificity of the primers designed. All the strains are listed in Table 1.

Bacteria were grown in MRS medium (

Identification of the strains isolated and detection of the intergenic-mob region of plasmid pF8801

A summary of the ropy strains isolated is presented in Table 1. Three morphological types were found: tetrad-forming cocci, chain-forming cocci and bacilli. All the isolates induced viscosity in liquid medium. P. damnosus and O. oeni isolates were identified using species-specific genomic probes as described by Lonvaud-Funel et al. (1991). The strains of P. damnosus IOEB0201 and IOEB0202 hybridized with the P. damnosus ATCC25248 total genomic probe, indicating that they belong to the species P.

Discussion

Lactic acid bacteria of the genera Lactococcus, Streptococcus, Leuconostoc, Lactobacillus and Pediococcus can produce exopolysaccharides in various media (Cerning, 1990). Species of Streptococcus mucilaginous, Leuconostoc and Pediococcus have been isolated from ropy wines (Sponholz, 1992). Pediococci and lactobacilli induce viscosity in ciders (Dueñas et al., 1995, Fernández et al., 1995). In this study, ropy wines were found to contain strains from several species: P. damnosus (IOEB0201), O.

Conclusion

In this study, several new ropy strains of various species were isolated from cider and wine, and in particular, a ropy O. oeni strain was described for the first time. The glucan-specific antibodies specifically agglutinate the cells carrying gene dps whether P. damnosus or O. oeni cells. They are good candidates for antibody-based immunoassays to detect spoilage bacteria in fermented beverages (Whiting et al., 1999). Moreover, new primers for the rapid detection by PCR of glucan-producing

Acknowledgements

We thank E. García for his antibody gift and his expert advice. The financial support of the French “Ministère de la Recherche” and the CIVB (Conseil Interprofessionnel du Vin de Bordeaux) is gratefully acknowledged.

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