Oenococcus oeni is the acidophilic lactic acid bacterial species most frequently associated with malolactic fermentation of wine. Since the description of the species (formerly Leuconostoc oenos), characterization of indigenous strains and industrially produced cultures by diverse typing methods has led to divergent conclusions concerning the genetic diversity of strains. In the present study, a multilocus sequence typing (MLST) scheme based on the analysis of eight housekeeping genes was developed and tested on a collection of 43 strains of diverse origins. The eight targeted loci were successfully amplified and sequenced for all isolates. Only three to 11 different alleles were detected for these genes. The average nucleotide diversity also was rather limited (0.0011 to 0.0370). Despite this limited allelic diversity, the combination of alleles of each strain disclosed 34 different sequence types, which denoted a significant genotypic diversity. A phylogenetic analysis of the concatenated sequences showed that all strains form two well distinct groups of 28 and 15 strains. Interestingly, the same groups were defined by pulsed-field gel electrophoresis, although this method targets different genetic variations. A minimum spanning tree analysis disclosed very few and small clonal complexes. In agreement, statistical analyses of MLST data suggest that recombination events were important during O. oeni evolution and contributed to the wide dissemination of alleles among strains. Taken together, our results showed that MLST is more efficient than pulsed-field gel electrophoresis for typing O. oeni strains, and they provided a picture of the O. oeni population that explains some conflicting results previously obtained.

Oenococcus oeni strains are well-known for their considerable phenotypic variations in terms of tolerance to harsh wine conditions and malolactic activity. Genomic subtractive hybridization (SH) between two isolates with differing enological potentials was used to elucidate the genetic bases of this intraspecies diversity and identify novel genes involved in adaptation to wine. SH revealed 182 tester-specific fragments corresponding to 126 open reading frames (ORFs). A large proportion of the chromosome-related ORFs resembled genes involved in carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, and replication, recombination, and repair. Six regions of genomic plasticity were identified, and their analysis suggested that both limited recombination and insertion/deletion events contributed to the vast genomic diversity observed in O. oeni. The association of selected sequences with adaptation to wine was further assessed by screening a large collection of strains using PCR. No sequences were found to be specific to highly performing (HP) strains alone. However, there was a statistically significant positive association between HP strains and the presence of eight gene sequences located on regions 2, 4, and 5. Gene expression patterns were significantly modified in HP strains, following exposure to one or more of the common stresses in wines. Regions 2 and 5 showed no traces of mobile elements and had normal GC content. In contrast, region 4 had the typical hallmarks of horizontal transfer, suggesting that the strategy of acquiring genes from other bacteria enhances the fitness of O. oeni strains.

Abstract;D T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that VD2-negative ( neg ) ;D T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of VD2 neg clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of VD2 neg clones, in contrast to VD2 + cells, prevented the development of HT29 tumors. VD2 neg clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1D and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with VD2 neg clones delayed the growth of HT29 s.c. tumors. The effect of in vivo ;D T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. ;D T-cell passive immunotherapy was dependent on the cytotoxic activity of the ;D effectors toward their targets because VD2 neg clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific VD2 neg cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy. [Cancer Res 2009;69(9):3971-8]