Diversity and enzymatic profiles of indigenous yeasts isolated from three types of palm wines produced in Côte d'Ivoire.

AIMS: To investigate the genotypic diversity and enzymatic activity of yeast flora isolated from spontaneous fermenting saps of various palm trees (Borassus aethiopum, Raphia hookeri, Elaeis guineensis) tapped for palm wines. METHODS AND RESULTS: PCR-restriction fragment length polymorphism of ITS-5.8S rDNA combined to 26S rRNA gene and/or the partial ACT1 gene sequencing were applied for yeast characterization, and their enzymatic profiles assessed by using API ZYM kits. Thirteen genera and 23 species were identified, with the highest diversity (14 species) in raffia wine. Saccharomyces cerevisiae was dominant and common to all palm wines. Some potentially pathogenic yeasts were also isolated. The majority of tested strains displayed high amylo-peptidase, phosphatase, ß-glucosidase and α-glucosidase activities and esterase activity. CONCLUSIONS: Diverse yeast species colonized palm wines, among which some were related to a specific type of wine and the majority of them have the ability to digest starch, sugar, protein or lipid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a first step in understanding the significance of indigenous yeast flora of palm wines from Côte d'Ivoire. This knowledge is important as a tool for establishing new indigenous yeast collection; which could be used for the product quality improvement and as enzyme sources for biotechnological purposes.

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial ß -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Ecological success of a group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrids in the northern european wine-making environment.

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δµ)(2) genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates.

Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica.

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode an extracellular lipase. The growth of a Deltalip2 strain on triglycerides as sole carbon source suggest an alternative pathway for triglycerides utilisation in this yeast. Here, we describe the isolation and the characterisation of the LIP7 and LIP8 genes which were found to encode a 366 and a 371-amino acid precursor protein, respectively. These proteins which belong to the triacylglycerol hydrolase family (EC 3.1.1.3) presented a high homology with the extracellular lipase CdLIP2 and CdLIP3 from Candida deformans. The physiological function of the lipase isoenzymes was investigated by creating single and multi-disrupted strains. Lip7p and Lip8p were found to correspond to active secreted lipases. The lack of lipase production in a Deltalip2 Deltalip7 Deltalip8 strain suggest that no additional extracellular lipase remains to be discovered in Y. lipolytica. The substrate specificity towards synthetic ester molecules indicates that Lip7p presented a maximum activity centred on caproate (C6) while that of Lip8p is in caprate (C10).

Sequence analysis of three mitochondrial DNA molecules reveals interesting differences among Saccharomyces yeasts.

The complete sequences of mitochondrial DNA (mtDNA) from the two budding yeasts Saccharomyces castellii and Saccharomyces servazzii, consisting of 25 753 and 30 782 bp, respectively, were analysed and compared to Saccharomyces cerevisiae mtDNA. While some of the traits are very similar among Saccharomyces yeasts, others have highly diverged. The two mtDNAs are much more compact than that of S.cerevisiae and contain fewer introns and intergenic sequences, although they have almost the same coding potential. A few genes contain group I introns, but group II introns, otherwise found in S.cerevisiae mtDNA, are not present. Surprisingly, four genes (ATP6, COX2, COX3 and COB) in the mtDNA of S.servazzii contain, in total, five +1 frameshifts. mtDNAs of S.castellii, S.servazzii and S.cerevisiae contain all genes on the same strand, except for one tRNA gene. On the other hand, the gene order is very different. Several gene rearrangements have taken place upon separation of the Saccharomyces lineages, and even a part of the transcription units have not been preserved. It seems that the mechanism(s) involved in the generation of the rearrangements has had to ensure that all genes stayed encoded by the same DNA strand.

Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization.

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

Only centromeres can supply the partition system required for ARS function in the yeast Yarrowia lipolytica.

Autonomously replicating sequences (ARSs) in the yeast Yarrowia lipolytica require two components: an origin of replication (ORI) and centromere (CEN) DNA, both of which are necessary for extrachromosomal maintenance. To investigate this cooperation in more detail, we performed a screen for genomic sequences able to confer high frequency of transformation to a plasmid-borne ORI. Our results confirm a cooperation between ORI and CEN sequences to form an ARS, since all sequences identified in this screen displayed features of centromeric DNA and included the previously characterized CEN1-1, CEN3-1 and CEN5-1 fragments. Two new centromeric DNAs were identified as they are unique, map to different chromosomes (II and IV) and induce chromosome breakage after genomic integration. A third sequence, which is adjacent to, but distinct from the previously characterized CEN1-1 region was isolated from chromosome I. Although these CEN sequences do not share significant sequence similarities, they display a complex pattern of short repeats, including conserved blocks of 9 to 14 bp and regions of dyad symmetry. Consistent with their A+T-richness and strong negative roll angle, Y. lipolytica CEN-derived sequences, but not ORIs, were capable of binding isolated Drosophila nuclear scaffolds. However, a Drosophila scaffold attachment region that functions as an ARS in other yeasts was unable to confer autonomous replication to an ORI-containing plasmid. Deletion analysis of CEN1-1 showed that the sequences responsible for the induction of chromosome breakage could be eliminated without compromising extrachromosomal maintenance. We propose that, while Y. lipolytica CEN DNA is essential for plasmid maintenance, this function can be supplied by several sub-fragments which, together, form the active chromosomal centromere. This complex organization of Y. lipolytica centromeres is reminiscent of the regional structures described in the yeast Schizosaccharomyces pombe or in multicellular eukaryotes.

The complete mitochondrial genome of yarrowia lipolytica.

We here report the complete nucleotide sequence of the 47.9 kb mitochondrial (mt) genome from the obligate aerobic yeast Yarrowia lipolytica. It encodes, all on the same strand, seven subunits of NADH: ubiquinone oxidoreductase (ND1-6, ND4L), apocytochrome b (COB), three subunits of cytochrome oxidase (COX1, 2, 3), three subunits of ATP synthetase (ATP6, 8 and 9), small and large ribosomal RNAs and an incomplete set of tRNAs. The Y. lipolytica mt genome is very similar to the Hansenula wingei mt genome, as judged from blocks of conserved gene order and from sequence homology. The extra DNA in the Y. lipolytica mt genome consists of 17 group 1 introns and stretches of A+Trich sequence, interspersed with potentially transposable GC clusters. The usual mould mt genetic code is used. Interestingly, there is no tRNA able to read CGN (arginine) codons. CGN codons could not be found in exonic open reading frames, whereas they do occur in intronic open reading frames. However, several of the intronic open reading frames have accumulated mutations and must be regarded as pseudogenes. We propose that this may have been triggered by the presence of untranslatable CGN codons. This sequence is available under EMBL Accession No. AJ307410.

DNA probes specific for the yeast species Debaryomyces hansenii: useful tools for rapid identification.

We developed a rapid and sensitive identification method for the halotolerant yeast Debaryomyces hansenii, based on the hybridization of species-specific sequences. These sequences were first identified in a survey of D. hansenii strains by random amplification of polymorphic DNA (RAPD) as giving conserved bands in all isolates tested. Two such conserved RAPD products, termed F01pro and M18pro, were cloned from the type strain CBS 767. The specificity of these probes was assessed by hybridizing them to DNA from various species of yeasts commonly found in cheese. F01pro and M18pro hybridized to the DNA of all D. hansenii var. hansenii tested, but not to DNA of other yeast species including the closely related strain of D. hansenii var. fabryii CBS 789. Hybridization patterns of F01pro and M18pro on digested genomic DNA of D. hansenii indicated that the sequences were repeated in the genome of all D. hansenii var. hansenii tested, and gave distinct polymorphic patterns. The single F01pro probe generated 11 different profiles for 24 strains by restriction fragment length polymorphism, using one restriction enzyme. F01pro represents a new type of repeated element found in fungi, useful for both identification and typing of D. hansenii and, together with M18pro, simplifies the study of this species in complex flora.

A Mycobacterium strain with extended capacities for degradation of gasoline hydrocarbons.

A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2, 4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.

Homologous recombination and transposition generate chromosome I neopolymorphism during meiosis in Saccharomyces cerevisiae.

We have studied the meiotic segregation of a chromosome length polymorphism (CLP) in the yeast Saccharomyces cerevisiae. The neopolymorphism frequently observed within the smallest chromosomes (I, VI, III and IX) is not completely understood. We focused on the analysis of the structure of chromosome I in 88 segregants from a cross between YNN295 and FL100trp. Strain FL100trp is known to carry a reciprocal translocation between the left arm of chromosome III and the right arm of chromosome I. PCR and Southern hybridization analyses were performed and a method for the rapid detection of chromosome I rearrangements was developed. Seven chromosome I types were identified among the 88 segregants. We detected 22 recombination events between homologous chromosomes I and seven ectopic recombination events between FL100trp chromosome III and YNN295 chromosome I. These recombination events occurred in 20 of the 22 tetrads studied (91%). Nine tetrads (41%) showed two recombination events. This showed that homologous recombination involving polymorphic homologues or heterologous chromosomes is the main source of neopolymorphism. Only one of the seven chromosome I variants resulted from a transposition event rather than a recombination event. We demonstrated that a Tyl element had transposed within the translocated region of chromosome I, generating mutations in the 3' LTR, at the border between U5 and PBS.

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Genomic exploration of the hemiascomycetous yeasts: 5. Saccharomyces bayanus var. uvarum.

Saccharomyces bayanus var. uvarum investigated here is the species closest to Saccharomyces cerevisiae. Random sequence tags (RSTs) allowed us to identify homologues to 2789 open reading frames (ORFs) in S. cerevisiae, ORFs duplicated in S. uvarum but not in S. cerevisiae, centromeres, tRNAs, homologues of Ty1/2 and Ty4 retrotransposons, and a complete rDNA repeat. Only 13 RSTs seem to be homologous to sequences in other organisms but not in S. cerevisiae. As the synteny between the two species is very high, cases in which synteny is lost suggest special mechanisms of genome evolution. The corresponding RSTs revealed that S. uvarum can exist without any S. cerevisiae DNA introgression. Accession numbers are from AL397139 to AL402278 in the EMBL databank.

Genomic exploration of the hemiascomycetous yeasts: 6. Saccharomyces exiguus.

Random sequence tags were obtained from a genomic DNA library of Saccharomyces exiguus. The mitochondrial genome appeared to be at least 25.7 kb in size, with a different organization compared to Saccharomyces cerevisiae. An unusual putative 953 bp long terminal repeated element associated to Ty3 was found. A set of 1451 genes was identified homologous to S. cerevisiae open reading frames. Only five genes were identified outside the S. cerevisiae taxon, confirming that S. exiguus is phylogenetically closely related to S. cerevisiae. Unexpectedly, numerous duplicated genes were found whereas they are unique in S. cerevisiae. The sequences are deposited at EMBL under the accession numbers: AL407377-AL409955.

Genomic exploration of the hemiascomycetous yeasts: 7. Saccharomyces servazzii.

The genome of Saccharomyces servazzii was analyzed with 2570 random sequence tags totalling 2.3 Mb. BLASTX comparisons revealed a minimum of 1420 putative open reading frames with significant homology to Saccharomyces cerevisiae (58% aa identity on average), two with Schizosaccharomyces pombe and one with a human protein, confirming that S. servazzii is closely related to S. cerevisiae. About 25% of the S. servazzii genes were identified, assuming that the gene complement is identical in both yeasts. S. servazzii carries very few transposable elements related to Ty elements in S. cerevisiae. Most of the mitochondrial genes were identified in eight contigs altogether spanning 25 kb for a predicted size of 29 kb. A significant match with the Kluyveromyces lactis linear DNA plasmid pGKL-1 encoded RF4 killer protein suggests that a related plasmid exists in S. servazzii. The sequences have been deposited with EMBL under the accession numbers AL402279-AL404848.

Genomic exploration of the hemiascomycetous yeasts: 9. Saccharomyces kluyveri.

The genome of Saccharomyces kluyveri was explored through 2528 random sequence tags with an average length of 981 bp. The complete nuclear ribosomal DNA unit was found to be 8656 bp in length. Sequences homologous to retroelements of the gypsy and copia types were identified as well as numerous solo long terminal repeats. We identified at least 1406 genes homologous to Saccharomyces cerevisiae open reading frames, with on average 58.1% and 72.4% amino acid identity and similarity, respectively. In addition, by comparison with completely sequenced genomes and the SwissProt database, we found 27 novel S. kluyveri genes. Most of these genes belong to pathways or have functions absent from S. cerevisiae, such as the catabolic pathway of purines or pyrimidines, melibiose fermentation, sorbitol utilization, or degradation of pollutants. The sequences are deposited in EMBL under the accession numbers AL404849-AL407376.

Genomic exploration of the hemiascomycetous yeasts: 14. Debaryomyces hansenii var. hansenii.

By analyzing 2830 random sequence tags (RSTs), totalling 2.7 Mb, we explored the genome of the marine, osmo- and halotolerant yeast, Debaryomyces hansenii. A contig 29 kb in length harbors the entire mitochondrial genome. The genes encoding Cox1, Cox2, Cox3, Cob, Atp6, Atp8, Atp9, several subunits of the NADH dehydrogenase complex 1 and 11 tRNAs were unambiguously identified. An equivalent number of putative transposable elements compared to Saccharomyces cerevisiae were detected, the majority of which are more related to higher eukaryote copia elements. BLASTX comparisons of RSTs with databases revealed at least 1119 putative open reading frames with homology to S. cerevisiae and 49 to other genomes. Specific functions, including transport of metabolites, are clearly over-represented in D. hansenii compared to S. cerevisiae, consistent with the observed difference in physiology of the two species. The sequences have been deposited with EMBL under the accession numbers AL436045-AL438874.

Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae.

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs in S. cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome of S. cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S. cerevisiae with itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.