Pediococci are found in foods and on plants and as beer-spoilage agents. The goal of the present study was to use the DNA sequences of the first three variable regions of the 165 rRNA gene, the 16S-23S rRNA internally transcribed spacer region sequence and approximately a third of the 60 kDa heat-shock protein gene to elucidate phylogenetic groupings within the genus Pediococcus. Phylogenetic trees were created with sequence data from 31 Pediococcus and three Lactobacillus isolates. Complete 16S rRNA gene sequences from selected Pediococcus isolates were also examined. The results were interpreted in relation to the currently accepted Pediococcus species. We found that, where previously done, speciation of many Pediococcus isolates is inaccurate. Also, one grouping of seven isolates did not include any currently recognized Pediococcus species type isolate. Our phylogenetic analyses support the conclusion that these seven isolates, all of brewing spoilage origin, belong to a novel species, for which the name Pediococcus claussenii sp. nov. is proposed (type strain P06(T0 = ATCC BAA-344(T) = DSM 14800(T)). Phylogenetic analysis has therefore helped to resolve problems surrounding species identification of Pediococcus isolates.

Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

To determine the locations and sequences of pediocin AcH production genes in Pediococcus parvulus ATO77 from vegetables, Lactobacillus plantarum WHE92 from Muenster cheese, and a lactose-fermenting isolate Pediococcus pentosaceus S34 from buffalo milk. Plasmid curing, Southern blot hybridization, and DNA sequence analysis indicate that pediocin AcH production genes are encoded by highly similar operons in unique plasmids designated pATO77 from P. parvulus ATO77, pS34 from P. pentosaceus S34, and pWHE92 from Lact. plantarum WHE92. Structure, immunity and secretion system genes are linked together in the operons, and the promoter sequences are the same. The amino acid sequences of the encoded proteins are highly conserved between plasmids. Pediocin AcH production genes are located within a plasmid-borne operon cassette in all lactic acid bacterial strains examined to date. All four genes needed for production are present within a single plasmid in each strain. This is the first demonstration that the expression of a class IIa bacteriocin is directed by a common gene cassette that has been disseminated to unique plasmids in different genera of lactic acid bacteria. These plasmids should be useful for expressing pediocin AcH in Pediococcus and Lactobacillus strains used in food production.

Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.

We have isolated three lactic acid bacteria (Lactobacillus suebicus CUPV221, Pediococcus parvulus CUPV1 and P. parvulus CUPV22) that produced high levels of 2-substituted (1,3)-beta-D-glucans which increased the viscosity of the growth media. The (1,3)-beta-D-glucan consisted of two main molecular species, with masses of approximately 10(7) and 10(4) Da, whose proportions varied among the strains. The three strains survived exposure to saliva and simulated gastric conditions at pH 5, with P. parvulus CUPV22 surviving at pH 3.1, and L. suebicus CUPV221 surviving at pH 1.8. All strains were resistant to pancreatin and bile salts. P. parvulus CUPV22 exhibited the highest adhesion (10.5%) to Caco-2 cells, which decreased to 1.2% after washing the cells. Finally, P. parvulus CUPV22 and L. suebicus CUPV221 induced the production of inflammation-related cytokines by polarized macrophages, and interestingly, L. suebicus stimulated the production of cytokine IL-10. These results indicate that the three strains have potential utility for the production of functional foods.

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.

Pediococcus parvulus 2.6 secretes a 2-substituted (1,3)-�]-D-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the P. parvulus 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (gutFRMCBA), whose products should be involved in sorbitol utilization and could generate substrates for UDP-glucose synthesis. Southern blot hybridization analysis showed that the cluster is located in a plasmid. Analysis of metabolic fluxes and production of the exopolysaccharide revealed that: (i) P. parvulus 2.6 is able to metabolize sorbitol, (ii) sorbitol utilization is repressed in the presence of glucose and (iii) sorbitol supports the synthesis of 2-substituted (1,3)-�]-D-glucan. The sorbitol cluster encodes two putative regulators, GutR and GutM, in addition to a phosphoenolpyruvate-dependent phosphotransferase transport system and sorbitol-6-phosphate dehydrogenase. Therefore, we investigated the involvement of GutR and GutM in the expression of gutFRMCBA. The promoter-probe vector pRCR based on the mrfp gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (P gut) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: Lactobacillus plantarum and Lactobacillus casei. Upon growth in the presence of sorbitol, but not of glucose, only the presence of P gut was required to support expression of mrfp in L. plantarum. In L. casei the presence of sorbitol in the growth medium and the pediococcal gutR or gutR plus gutM in the genome was required for P gut functionality. This demonstrates that: (i) P gut is required for expression of the gut cluster, (ii) P gut is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, trans-complementation for activation of P gut exists in L. plantarum but not in L. casei.