Oenococcus oeni is responsible for the malolactic fermentation of wines. Genomic diversity has been recently established in the species and extensive attention is now being given to the genomic bases of strain-specific differences. We explored the role of insertion sequences (IS), which are considered as driving forces for novel genotypic and phenotypic variants in prokaryotes. The present study focuses on members of the IS30 family, which are widespread among lactic acid bacteria. An in silico analysis of the three available genomes of O. oeni in combination with the use of an inverse PCR strategy targeting conserved IS30-related sequences indicated the presence of seven IS30 copies in the pangenome of O. oeni. A primer designed to anneal to the conserved 3' end of the IS30 element was paired with each of the seven primers selected to bind to unique sequences upstream of each of the seven mobile elements identified. The study presents an overview of the abundance, and the genomic environment of IS30 elements in the O. oeni pangenome and shows that the two existing genetic sub-populations previously described in the species through multilocus sequence typing analysis (MLST) differ in their IS30 content. Possible IS30 impacts on bacterial adaptation are discussed.

ABSTRACT 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.

Oenococcus oeni is a minor species among the lactic acid bacteria naturally occurring on grape fruits, but it rapidly becomes predominant in wine and is generally the only detectable species during malolactic fermentation. To identify traits responsible for this so efficient adaptation to the wine environment, we used a panel of genomic approaches to examine O. oeni at both species and infra-species levels. Strain typing methods revealed a huge diversity of strains whose genomes are shaped by recombination events and that form at least two well-defined genetic groups. Evidences for genomic plasticity and horizontal gene transfers were confirmed by genome sequence comparisons and by subtractive genomic hybridizations. Differential DNA regions specific of some strains code for enzymes that may help bacteria to cope with the harsh conditions of wine. Taken together our works support the previously suggested idea that O. oeni is a fast-evolving species and they provide the first clues about how some strains can better survive in wine. In addition they make it possible for the first time to select the best adapted strains for industrial applications on the basis of genetic traits rather than phenotypic criteria.

Genomic 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.

Taking advantage of recent technology developments of 454 pyrosequencing we have undertaken whole genome sequencing of O. oeni strain IOEB 8413. This strain performs malolactic fermentation, but has a relatively low level of adaptation to oenological environment. The genome draft was obtained with Genome Sequencer FLX System (Roche). The generated 111 Mb of raw data (406 255 individual reads) was processed with Newbler Assembler through two different modes. The assembly resulting from the de novo mode consisted of 39 major contigs attaining an average 50x coverage. Manual draft inspection revealed that, independently of the number of contigs, it contained only 19 real gaps. Cumulated size of missing fractions was estimated to be less than 10 kb, thus allowing us one-step finishing by Sanger sequencing. For the second assembly mode, particularly indicated for resequencing projects, we have used as a reference the recently achieved genome of strain ATCC BAA-1163 (LAB9, communication Bon et al.). The 1,8-Mb chromosome is covered by 90% of all mapped reads, whereas the fully covered 3,9-kb high-copy plasmid monopolizes the remaining 10%. More than 97% of all reads were fully mapped to the reference (chromosome or plasmid) and organized in 18 mapping contigs. Less than 2% of reads aligned partially at the oriC linearization site or corresponded to repeat regions (rRNA operons). Only 429 reads are unique meaning that both strains have very close nucleotide content. Preliminary evaluation of the two genomes indicates no significant large-scale rearrangements. Functional annotation as well as further comparative analyses between available strains (including PSU-1) will await completion of the strain IOEB 8413 genome.

Growth of the lactic acid bacterium Oenococcus oeni under hyperosmotic constraint was investigated in a chemically defined medium. The bacterium could grow on media with an elevated osmolality, preferably below 1.5 Osm kg(-)(1) H(2)O. At osmolalities comprised between 0.6 and 1.5 Osm kg(-)(1) H(2)O, the growth deficit elicited by the sugars glucose and fructose was slightly more severe than with salts (NaCl or KCl). In contrast to what was observed in other lactic acid bacteria, proline, glycine betaine and related molecules were unable to relieve inhibition of growth of O. oeni under osmotic constraint. This was correlated to the absence of sequences homologous to the genes coding for glycine betaine and/or proline transporters described in Lactococcus lactis and Lactobacillus plantarum. The amino acid aspartate proved to be osmoprotective under electrolyte and non-electrolyte stress. Examination of the role of peptides during osmoregulation showed that proline- and glutamate-containing peptides were protective under salt-induced stress, and not under sugar-induced stress. Under high salt, PepQ a cytoplasmic prolidase that specifically liberated proline from di-peptides increased activity, while PepX (X-prolyl-dipeptidyl aminopeptidase) and PepI (iminopeptidase) activities were unaffected. Our data suggest that proline- and glutamate-containing peptides may contribute to the adaptation of O. oeni to high salt through their intracellular hydrolysis and/or direct accumulation.

La bacterie lactique Oenococcus oeni est consideree comme le principal microorganisme responsable de la fermentation malo-lactique (FML), qui diminue l'acidite totale et augmente la qualite et la stabilite des vins. O. oeni est presente en faible quantite dans le mout, avant et pendant la fermentation alcoolique. La bacterie se developpe dans le vin lorsque les sucres ont ete completement fermentes. Le succes de cette seconde fermentation, cruciale pour le bon deroulement de la vinification, depend de l'aptitude de O. oeni a faire face a un environnement hostile. Les problemes d'induction de FML sont classiquement lies aux effets inhibiteurs cumulatifs du pH acide, de la teneur elevee en alcool et de la presence de differents composes (acides gras, acides phenoliques et tanins). Aussi, pour pallier l'eventuelle deficience des souches indigenes de O. oeni a initier la FML, certains professionnels inoculent en fin de fermentation alcoolique des levains commerciaux. Malgre les progres realises, les resultats restent aleatoires. La performance des levains repose a la fois sur la selection des souches et la qualite de la preparation industrielle, qui sont encore mal maitrisees. L'une et l'autre beneficieront d'une meilleure comprehension (i) des mecanismes responsables de la tolerance de O. oeni au milieu vin (acidite, ethanol, froid ...) et (ii) de la regulation de la reponse au stress chez cette bacterie (Mills et al., 2005). Parmi les contraintes non abordees a ce jour, la tolerance aux variations de pression osmotique est un parametre important. De telles variations existent lorsque les cellules se trouvent dans des conditions de faible activite de l'eau. C'est le cas pendant la fabrication des preparations lyophilisees. Elles interviennent egalement lorsque la bacterie subit des phases de dessication sur les baies ou sur les differents equipements des chais, ou encore au debut de la vinification lorsque les mouts se caracterisent par leur forte teneur en sucres. La croissance de Oenococcus oeni sous contrainte hyperosmotique a ete etudiee dans un milieu chimiquement defini. La bacterie se developpe sur des milieux d'osmolalite elevee, au dessous de 1.5 osmoles kg-1 H2O. Pour des osmolalites comprises entre 0.6 et 1.5 osmoles kg-1 H2O, le deficit de croissance releve en presence de sucres est plus important qu'en presence de sels. Differentes familles de molecules a proprietes osmoprotectrices ont ete testees. Contrairement a ce qui a ete decrit chez d'autres bacteries lactiques, la proline, la glycine betaine et les composes proches ne peuvent restaurer la croissance de O. oeni en situation de contrainte osmotique. Cette observation a ete correlee a l'absence de sequences homologues aux genes codant les transporteurs de glycine betaine et/ou de proline decrits chez Lactococcus lactis et Lactobacillus plantarum. L'acide amine aspartate s'est revele protecteur en contrainte electrolyte et non electrolyte. D'autre part, l'evaluation du role de differents petits peptides (Piuri et al., 2003) a montre un effet protecteur de di-peptides contenant la proline ou le glutamate Cette protection est uniquement observee lors d'un stress de nature electrolyte. Le phenomene est lie a une accumulation directe et/ou une degradation intra-cellulaire.

Using molecular techniques and sequencing, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium involved in red winemaking. A relationship between the phenotypic and genotypic characterization of 16 O. oeni strains isolated from wine with different levels of enological potential was shown. The study was based on the comparative genomic analysis by subtractive hybridization between two strains of O. oeni with opposite enological potential. The genomic sequences obtained from subtractive hybridization were amplified by polymerase chain reaction and sequenced for the 16 strains. A considerable diversity among strains of O. oeni was observed.

Oenococcus oeni is the best adapted bacterium to the harsh wine medium. Its growth is necessary after the alcoholic fermentation for the improvement of wine during malolactic fermentation. Inspite of the low pH, ethanol concentration and several other bacterial inhibitors, such as free fatty acids and some phenolic acids, O. oeni actively grow in wine. The efficiency of different starter cultures often used during the winemaking process is related to their inherent ability to survive and multiply. Bacteria use sophisticated mechanisms to enable them to survive a variety of environmental stresses. Among the stress genes whose expression changes in response to various stimuli in Oenococcus oeni several have already been identified. The objective of the present study was to determine genetic markers which should be used for the selection of O. oeni strains. A comparative genomic analysis was carried out between two O. oeni strains with opposite oenological potential. By Polymerase Chain Reaction (PCR) we checked among 80 O. oeni strains the presence of 46 genomic sequences derived from the comparative genomic analysis. Statistical analysis revealed 11 possible genetics markers. Their expression was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR), for 14 efficient O. oeni strains and 5 less adapted. The quantification of transcription levels of the 11 genetics markers after a heat shock, acid and ethanol stress, exponential and stationary phase was determined. According to the gene, their transcription levels increased or decreased greatly in stress conditions.

The Comprehensive Yeast Genome Database (CYGD) compiles a comprehensive data resource for information on the cellular functions of the yeast Saccharomyces cerevisiae and related species, chosen as the best understood model organism for eukaryotes. The database serves as a common resource generated by a European consortium, going beyond the provision of sequence information and functional annotations on individual genes and proteins. In addition, it provides information on the physical and functional interactions among proteins as well as other genetic elements. These cellular networks include metabolic and regulatory pathways, signal transduction and transport processes as well as co-regulated gene clusters. As more yeast genomes are published, their annotation becomes greatly facilitated using S.cerevisiae as a reference. CYGD provides a way of exploring related genomes with the aid of the S.cerevisiae genome as a backbone and SIMAP, the Similarity Matrix of Proteins. The comprehensive resource is available under http://mips.gsf.de/genre/proj/yeast/.

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5' end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns' features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.