Identification of pOENI-1 and related plasmids in Oenococcus oeni strains performing the malolactic fermentation in wine.

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine

Multilocus Sequence Typing of Oenococcus oeni: Detection of Two Subpopulations Shaped by Intergenic Recombination▿ †

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

Stress-driven method bio-inspired by long bone structure for mechanical part mass reduction by removing geometry at macro and cell-unit scales

International audienceMass reduction is a main issue in mechanical design. Over millions of years, Nature had to face this issue.Nature came up with an efficient solution using a stress-driven structure to reduce the mass of bones whilesaving their mechanical performances. This optimized structure is used in several species and persiststhroughout Evolution. Thus, it may be considered as optimal for this issue. In this article, a method bio-inspired from both bone medullar cavity and trabecular structure is proposed to reduce the mass of partssubjected to mechanical stresses. The objective of this method is to provide high mass reduction, just likebone does. First, the method removes iteratively unloaded areas of material from the mechanical part tomimic the medullar cavity structure. Second, a final mass reduction is done integrating small holes bio-inspired from trabecular structure in the remaining material. An experimental validation was carried out on atorsion disc and provided a 60% mass reduction. Using this mass reduction rate, the topology optimizationmethod was used to define a standard geometry to evaluate the mechanical performances of the proposedmethod. Experimental results highlight that regarding torsional stiffness, the bio-inspired part is 27% stifferthan the standard on

Performance domains of bio-inspired and triangular lattice patterns to optimize the structures’ stiffness

Mass reduction of mechanical systems is a recurrent objective in engineering, which is often reached by removing material from its mechanical parts. However, this material removal leads to a decrease of mechanical performances for the parts, which must be minimized and controlled to avoid a potential system failure. To find a middle-ground between material removing and mechanical performances), material must be kept only in areas where it is necessary, for example using stress-driven material removal methods. These methods use the stress field to define the local material removal based on two local parameters: the local volume fraction vf and the structural anisotropy orientation β. These methods may be based on different types of cellular structure patterns: lattice-based or bio-inspired. The long-term objective of this study is to improve the performance of stress-driven methods by using the most efficient pattern. For this purpose, this study investigates the influence of vf and β on the mechanical stiffness of three planar cellular structures called Periodic Stress-Driven Material Removal (PSDMR) structures. The first, taken from the literature, is bio-inspired from bone and based on a square pattern. The second, developed in this study, is also bio-inspired from bone but based on a rectangular pattern. The third is a strut-based lattice pattern well documented in the literature for its isotropic behavior. These three patterns are compared in this study in terms of relative longitudinal stiffness, obtained through linear elastic compressive tests by finite element analysis. It is highlighted that each PSDMR pattern has a specific domain in which it performs better than the two others. In future works, these domains could be used in stress-driven material removal methods to select the most adequate pattern or a mix of them to improve the performances of parts

Primers list.

a<p>Primers used in qPCR assays.</p>b<p>Product sizes obtained for pOENI-1 and pOENI-1v2.</p

Characterization of an acquired dps-containing gene island in the lactic acid bacterium Oenococcus oeni

International audienceGenomic surveillance of the two available genome sequences from Oenococcus oeni indicated the presence of a 480-bp ORF, encoding a 18.7-kDa protein named DpsA, whose deduced amino acid sequence was in significant homology with the Dps proteins from Escherichia coli and various lactic acid bacteria. Consistent with the role of Dps proteins as a key component in response to oxydative stress, the cloned dpsA gene complemented the dps- mutant of E. coli and conferred resistance to hydrogen peroxide. More interestingly, the oenococcal gene also protected E. coli from the deleterious effects of wine, copper and ferric ions, three stressors encountered by O. oeni in its environment. The dpsA gene was flanked by IS-related elements. The entire region was characterized by an anomalously high GC content compared to those reported for complete PSU-1 and ATCC-BAA-1163 oenococcal genomes. In the O. oeni species, the dpsA gene was present in 15 of the 38 tested isolates. Positive strains were apparently unrelated, as they originated from different geographical areas and types of wines. No change in tolerance to wine was observed between strains harbouring dpsA and those not harbouring this gene. Our results suggest that some O. oeni have acquired the dpsA gene as part of a mobile element. DpsA probably increases the O. oeni fitness in response to environmental challenges. However, the physiological condition under which it adds a selective advantage to O. oeni during winemaking remains to be found

Distribution of pOENI-1 genes in 44 <i>O. oeni</i> strains.

<p>The dendrogram was constructed from DNA banding patterns obtained by NotI-PFGE analysis of 44 <i>O. oeni</i> strains. <i>Oenococcus kitaharae</i> was used as outgroup. Strain S11 was positioned on the basis of MLST data since no NotI-PFGE pattern was obtained for this strain. The presence (filled square) or absence (empty squares) of plasmid genes <i>repA</i>, <i>tauE</i>, <i>oye</i> and of the chromosomal gene OEOE_0812 were determined by PCR. The presence/absence of a region encompassing the <i>oye</i> and <i>parB</i> genes was also investigated. IOEB: Institute of oenology of Bordeaux, S: SARCO, ATCC: American type culture collection. Indutrial strains are marked with asterisks. Letters A and B in the dendrogram represent two phylogenetic groups of strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049082#pone.0049082-Bilhere1" target="_blank">[36]</a>.</p

Comparison of growth in wine of isogenic strains with/without plasmids.

<p>Kinetics of alcoholic fermentation (CO₂ released, dark line), MLF (colored solid lines) and bacterial populations (colored dotted lines) were monitored in a sterile grape must inoculated with industrial wine yeasts and 10³.ml⁻¹ bacteria carrying pOENI-1 or pOENI-1v2 (red lines), bacteria without plasmids (blue lines) or a mixture of both (green lines). Kinetics of AF (dark symbols) is the mean of the three experiments.</p

<i>O. oeni</i> strains used in this study.

a<p>IOEB: Institute of oenology of Bordeaux, S: SARCO, ATCC: American type culture collection.</p