Oenococcus oeni genome plasticity is associated with fitness.

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.

Tolerance to high osmolality of the lactic acid bacterium Oenococcus oeni and identification of potential osmoprotectants.

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.

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns.

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.

HIV-1 integrase interacts with yeast microtubule-associated proteins.

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.

IS30 elements are mediators of genetic diversity in Oenococcus oeni.

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.

Intraspecific diversity of Oenococcus oeni strains determined by sequence analysis of target genes.

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.

Ylli, a non-LTR retrotransposon L1 family in the dimorphic yeast Yarrowia lipolytica.

During the course of a random sequencing project of the genome of the dimorphic yeast Yarrowia lipolytica, we have identified sequences that were repeated in the genome and that matched the reverse transcriptase (RT) sequence of non-long terminal repeat (non-LTR) retrotransposons. Extension of sequencing on each side of this zone of homology allowed the definition of an element over 6 kb long. The conceptual translation of this sequence revealed two open reading frames (ORFs) that displayed several characteristics of non-LTR retrotransposons: a Cys-rich motif in the ORF1, an N-terminal endonuclease, a central RT, and a C-terminal zinc finger domain in the ORF2. We called this element Ylli (for Y. lipolytica LINE). A total of 19 distinct repeats carrying the 3' untranslated region (UTR) and all ending with a poly-A tail were detected. Most of them were very short, 17 being 134 bp long or less. The number of copies of Ylli was estimated to be around 100 if these short repeats are 5' truncations. No 5' UTR was clearly identified, indicating that entire and therefore active elements might be very rare in the Y. lipolytica strain tested. Ylli does not seem to have any insertion specificity. Phylogenetic analysis of the RT domain unambiguously placed Ylli within the L1 clade. It forms a monophyletic group with the Zorro non-LTR retrotransposons discovered in another dimorphic yeast Candida albicans. BLAST comparisons showed that ORF2 of Ylli is closely related to that of the slime mold Dictyostelium discoideum L1 family, TRE.

Genomic evolution of the long terminal repeat retrotransposons in hemiascomycetous yeasts.

We identified putative long terminal repeat- (LTR) retrotransposon sequences among the 50,000 random sequence tags (RSTs) obtained by the Génolevures project from genomic libraries of 13 Hemiascomycetes species. In most cases additional sequencing enabled us to assemble the whole sequences of these retrotransposons. These approaches identified 17 distinct families, 10 of which are defined by full-length elements. We also identified five families of solo LTRs that were not associated with retrotransposons. Ty1-like retrotransposons were found in four of five species that are phylogenetically related to Saccharomyces cerevisiae (S. uvarum, S. exiguus, S. servazzii, and S. kluyveri but not Zygosaccharomyces rouxii), and in two of three Kluyveromyces species (K. lactis and K. marxianus but not K. thermotolerans). Only multiply crippled elements could be identified in the K. lactis and S. servazzii strains analyzed, and only solo LTRs could be identified in S. uvarum. Ty4-like elements were only detected in S. uvarum, indicating that these elements appeared recently before speciation of the Saccharomyces sensu stricto species. Ty5-like elements were detected in S. exiguus, Pichia angusta, and Debaryomyces hansenii. A retrotransposon homologous with Tca2 from Candida albicans, an element absent from S. cerevisiae, was detected in the closely related species D. hansenii. A complete Ty3/gypsy element was present in S. exiguus, whereas only partial, often degenerate, sequences resembling this element were found in S. servazzii, Z. rouxii, S. kluyveri, C. tropicalis, and Yarrowica lipolytica. P. farinosa (syn. P. sorbitophila) is currently the only yeast species in which no LTR retrotransposons or remnants have been found. Thorough analysis of protein sequences, structural characteristics of the elements, and phylogenetic relationships deduced from these data allowed us to propose a classification for the Ty1/copia elements of hemiascomycetous yeasts and a model of LTR-retrotransposon evolution in yeasts.