Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Overlapping responses between salt and oxidative stress in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.

Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts.

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.

Yeast-like fungi and yeasts in withered grape carposphere: Characterization of Aureobasidium pullulans population and species diversity.

Yeast-like fungi and yeasts residing on carposphere of withered grapes for Italian passito wine production have been scarcely investigated. In the present study, isolates from single berries, both sound and damaged, of Nosiola, Corvina and Garganega varieties were analyzed at the end of the withering process. Great variation of cell concentration among single berries was observed. In sound berries, yeast-like fungi were significantly more frequent than yeasts. Species identification of isolates was carried out by BLAST comparative analysis on gene databases and phylogenetic approach. All yeast-like fungi isolates belonged to Aureobasidium pullulans. They displayed different culture and physiological characteristics and inhibitory capacity against phytopathogenic fungi. Moreover, PCR profile analysis revealed high genotypic similarity among these strains. A total of 35 species were recognized among yeast isolates. Ascomycetes prevailed over basidiomycetes. To the best of our knowledge, Naganishia onofrii and Rhodosporidiobolus odoratus were identified for the first time among yeasts isolated from grapes, must or wine. Hanseniaspora uvarum and Starmerella bacillaris were the most frequent species. Most species were found only in one grape variety (nine in Nosiola, 10 in Corvina and five in Garganega). The sanitary state of withered grapes could have an important impact on the structure of these epiphytic populations.

Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1­5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1­5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1­5 protein inside experimental data-based models showed how two chains of HAMP1­5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1­5 structure mediate its function.

Hanseniaspora nectarophila sp. nov., a yeast species isolated from ephemeral flowers.

Seven apiculate yeast strains that were isolated from the flowers of Syphocampylus corymbiferus Pohl in Brazil are genetically, morphologically and phenotypically distinct from recognized species of the genera Hanseniaspora and Kloeckera. Genetic discontinuities between the novel strains and their closest relatives were found using a networking approach based on the concatenated sequences of the rRNA gene (internal transcribed spacer and D1/D2 of the LSU), and the protein-coding genes for actin and translation elongation factor-1α. Phylogenetic analysis based on the rRNA and the actin gene placed the novel species represented by the strains in close relationship to Hanseniaspora meyeri and Hanseniaspora clermontiae. PCR fingerprinting with microsatellite primers confirmed the genetic heterogeneity of the novel species. The name Hanseniaspora nectarophila sp. nov. is proposed, with UFMG POG a.1(T) ( = ZIM 2311(T)  = CBS 13383(T)) as the type strain; MycoBank no. MB807210. As the current description of the genus does not allow the presence of multilateral budding, an emended diagnosis of the genus Hanseniaspora Zikes is proposed.

The microbiology of Bandji, palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts, lactic acid and acetic acid bacteria.

AIM: To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro-organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa. METHODS AND RESULTS: Physicochemical characterization was performed using conventional methods. Identification of micro-organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro-organisms were screened respectively by amplification of the ITS1-5.8S rDNA-ITS2/16S-23S rDNA ITS regions and repetitive sequence-based PCR (rep-PCR). The physicochemical characteristics of samples were: pH: 3.48-4.12, titratable acidity: 1.67-3.50 mg KOH g(-1), acetic acid: 0.16-0.37%, alcohol content: 0.30-2.73%, sugars (degrees Brix): 2.70-8.50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro-organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis. CONCLUSION: fermentation of palm sap from B. akeassii involved multi-yeast-LAB-AAB cultures at genus, species and intraspecies level. SIGNIFICANCE AND IMPACT OF THE STUDY: First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by-products.

Candida albicans--a pre-whole genome duplication yeast--is predominantly aerobic and a poor ethanol producer.

Yeast species belonging to the lineage that underwent the whole genome duplication (WGD), and including Saccharomyces cerevisiae, can grow under anaerobiosis and accumulate ethanol in the presence of glucose and oxygen. The pre-WGD yeasts, which branched from the S. cerevisiae lineage just before the WGD event, including Kluyveromyces lactis, are more dependent on oxygen and do not accumulate large amounts of ethanol in the presence of excess oxygen. Yeasts that belong to the so-called 'lower branches' of the yeast phylogenetic tree and diverged from S. cerevisiae more than 200 million years ago have so far not been thoroughly investigated for their physiology and carbon metabolism. Here, we have studied several isolates of Candida albicans and Debaryomyces hansenii for their dependence on oxygen. Candida albicans grew very poorly at an oxygen concentration <1 p.p.m. and D. hansenii could not grow at all. In aerobic batch cultivations, C. albicans exhibited a predominantly aerobic metabolism, accumulating only small amounts of ethanol (0.01-0.09 g g(-1) glucose). Apparently, C. albicans and several other pre-WGD yeasts still exhibit the original traits of the yeast progenitor: poor accumulation of ethanol under aerobic conditions and strong dependence on the presence of oxygen.

An optimized procedure for the enological selection of non-Saccharomyces starter cultures.

The apiculate yeasts are the species predominating the first stage of grape must alcoholic fermentation and are important for the production of desired volatile compounds. The aim of the present investigation was to establish a protocol for the enological selection of non-Saccharomyces strains directly isolated from a natural must fermentation during the tumultuous phase. At this scope, fifty Hanseniaspora uvarum isolates were characterized at strain level by employing a new combined PCR-based approach. One isolate representative of each identified strain was used in fermentation assays to assess strain-specific enological properties. The chemical analysis indicated that all the analyzed strains were low producers of acetic acid and hydrogen sulphide, whereas they showed fructophilic character and high glycerol production. Analysis of volatile compounds indicated that one strain could positively affect, during the alcoholic fermentation process, the taste and flavour of alcoholic beverages. The statistical evaluation of obtained results indicated that the selected autochthonous H. uvarum strain possessed physiological and technological properties which satisfy the criteria indicated for non-Saccharomyces wine yeasts selection. Our data suggest that the described protocol could be advantageously applied for the selection of non-Saccharomyces strains suitable for the formulation of mixed or sequential starters together with Saccharomyces cerevisiae.

Interactions among HAMP domain repeats act as an osmosensing molecular switch in group III hybrid histidine kinases from fungi.

The members of group III hybrid histidine kinases (HHK) are ubiquitous in fungi. Group III HHK have been implicated to function as osmosensors in the high osmolarity glycerol (HOG) pathway that is essential for fungal survival under high osmolarity stress. Recent literature suggests that group III HHK are also involved in conidia formation, virulence in several filamentous fungi, and are an excellent molecular target for antifungal agents. Thus, group III HHK constitute a very important group of sensor kinases. Structurally, group III HHK are distinct from Sln1p, the osmosensing HHK that regulates the HOG pathway in Saccharomyces cerevisiae. Group III HHK lack any transmembrane domain and typically contain HAMP domain repeats at the N terminus. Until now, it is not clear how group III HHK function as an osmosensor to regulate the HOG pathway. To investigate this, we undertook molecular characterization of DhNIK1, an ortholog from osmotolerant yeast Debaryomyces hansenii. We show here that DhNIK1 could complement sln1 mutation in S. cerevisiae thereby confirming its role as a bona fide osmosensor. We further investigated the role of HAMP domains by deleting them systematically. Our results clearly indicate that the HAMP4 domain is crucial for osmosensing by DhNik1p. Most importantly, we also show that the alternative interaction among the HAMP domains regulates the activity of DhNik1p like an "on-off switch" and thus provides, for the first time, an insight into the molecular mechanism of osmosensing by this group of HHKs.

[Exon-intron structure of genes of fungi genomes].

Objects of research--genes of A. fumigatus, C. glabrata, C. neoformans, D. hansenii, E. cuniculi, E. gossypii, K. lactis, M. grisea, N. crassa, S. cerevisiae, S. pombe, U. maydis and Y. lipolytica fungi genomes. Methods of research are computer calculation. The content of genes with exon-intron structure in fungi genomes are from 0.7 to 97.0%. The exon-intron gene structure was changed when the portion of genes with introns increased. In A. fumigatus, C. neoformans, M. grisea, N. crassa, S. pombe and U. maydis genomes that linear dependence between gene lengths, sum of exon lengths and intron number in genes was established.

Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis.

Doenjang is a traditional Korean fermented soybean paste that provides a major source of protein. The microbial diversity of 10 samples of doenjang (5 commercially manufactured products and 5 homemade products) was investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). In the first step, the nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers. Subsequently, these products were used as a template in a nested PCR to obtain fragments suitable for DGGE. The bacterial DGGE profile targeting the V3 region of the 16S rRNA gene indicated that lactic acid bacteria such as Leuconostoc mesenteroide, Tetragenococcus halophilus, and Enterococcus faecium were the predominant species. However, bands corresponding to Bacillus species, known to be the main organisms in doenjang, were not detected under the conditions described above. When selective PCR was conducted using a primer specific for Bacillus species, Bacillus subtilis and B. licheniformis were detected in several doenjang samples. In analysis of fungi, Mucor plumbeus, Aspergillus oryzae, and Debaryomyces hansenii were the most common species in the doenjang samples. On the basis of DGGE, a few differences in community structure were found for different samples. Also, cluster analysis of the DGGE profile revealed that the microbial diversity did not differ clearly between commercially manufactured and homemade products. The nested PCR-DGGE technique was used for the first time in this study to asses a microbial community in doenjang and proved to be effective in profiling microbial diversity.

Characterization of DhKHA1, a gene coding for a putative Na(+) transporter from Debaryomyces hansenii.

The KHA1 gene from Debaryomyces hansenii has been identified and characterized by heterologous expression in Saccharomyces cerevisiae. The gene is orthologous to ScKHA1, previously reported in S. cerevisiae, and on the basis of the deduced amino acid sequence, DhKha1p can be classified as an Na(+)/H(+) transporter. Reverse transcriptase (RT)-PCR experiments indicated that the expression level of DhKHA1 was not dependent on high pH or on the presence of a high salt level in the growth medium. Overexpression of DhKHA1 in a salt-sensitive S. cerevisiae mutant (ena1-4 nha1 kha1) rendered cells specifically more tolerant to Na(+). In addition, internal K(+) and Na(+) measurements and experiments performed with green fluorescence protein (GFP)-tagged DhKha1p indicated the intracellular localization of this protein when expressed in S. cerevisiae.

Why do some yeast species require niacin for growth? Different modes of NAD synthesis.

NAD holds a key position in metabolism and cellular regulatory events as a major redox carrier and a signalling molecule. NAD biosynthesis pathways have been reconstructed and compared in seven yeast species with completely sequenced genomes, including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and Schizosaccharomyces pombe. Both amino acid and nucleotide sequence similarity analysis in silico indicated that de novo NAD biosynthesis might not exist in K. lactis, C. glabrata and Schiz. pombe, while other species have the kynurenine pathway. It also showed that the NAD salvage pathway via nicotinic acid and nicotinic acid mononucleotide is conserved in all of these yeasts. Deletion of KlNPT1 (the gene for nicotinate phosphoribosyl-transferase) is lethal, which demonstrates that this salvage pathway, utilizing exogenous nicotinic acid, is the unique route to synthesize NAD in K. lactis. The results suggested that the basis of the variation of niacin requirements in yeasts lies in their different combinations of NAD biosynthesis pathways. The de novo pathway is absent but the salvage pathway is conserved in niacin-negative yeasts, while both pathways coexist in niacin-positive yeasts.

Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation.

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.

APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity.

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36-39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60 degrees C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis.

BACKGROUND: To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available. RESULTS: A dataset of 345,829 genes was extracted from 42 publicly available fungal genomes. Supertree methods were employed to derive phylogenies from 4,805 single gene families. We found that the average consensus supertree method may suffer from long-branch attraction artifacts, while matrix representation with parsimony (MRP) appears to be immune from these. A genome phylogeny was also reconstructed from a concatenated alignment of 153 universally distributed orthologs. Our MRP supertree and concatenated phylogeny are highly congruent. Within the Ascomycota, the sub-phyla Pezizomycotina and Saccharomycotina were resolved. Both phylogenies infer that the Leotiomycetes are the closest sister group to the Sordariomycetes. There is some ambiguity regarding the placement of Stagonospora nodurum, the sole member of the class Dothideomycetes present in the dataset. Within the Saccharomycotina, a monophyletic clade containing organisms that translate CTG as serine instead of leucine is evident. There is also strong support for two groups within the CTG clade, one containing the fully sexual species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. The second major clade within the Saccharomycotina contains species whose genomes have undergone a whole genome duplication (WGD), and their close relatives. We could not confidently resolve whether Candida glabrata or Saccharomyces castellii lies at the base of the WGD clade. CONCLUSION: We have constructed robust phylogenies for fungi based on whole genome analysis. Overall, our phylogenies provide strong support for the classification of phyla, sub-phyla, classes and orders. We have resolved the relationship of the classes Leotiomyctes and Sordariomycetes, and have identified two classes within the CTG clade of the Saccharomycotina that may correlate with sexual status.

Use of molecular methods for the identification of yeast associated with table olives.

A molecular approach is used for the identification of yeast isolated from table olives. Our results validate those obtained in the past by the classical biochemical methodology. Yeast were isolated from both aerobically and anaerobically processed black table olives and also from canned seasoned green table olives. Molecular identification methodology used included restriction pattern analysis of both PCR-amplified 5.8S rRNA gene and internal transcribed spacers ITS(1) and ITS(2). For some species, sequence analysis of the 26S rRNA gene was necessary. These techniques allowed the identification of three yeast species (Issatchenkia occidentalis, Geotrichum candidum and Hanseniaspora guilliermondii) which had not been described previously in table olives. Saccharomyces cerevisiae and Candida boidinii were the most frequent species in green seasoned olives and processed black olives, respectively. The molecular study of total DNA variability among the S. cerevisiae strains isolated indicates a quite heterogeneous population, with at least four different restriction patterns.

DhARO4, an amino acid biosynthetic gene, is stimulated by high salinity in Debaryomyces hansenii.

The highly halotolerant yeast Debaryomyces hansenii when grown in the presence of 2M NaCl, increased the expression of ARO4 which is involved in the biosynthesis of aromatic amino acids. The function of the isolated gene was verified by complementation of a Saccharomyces cerevisiae null mutant, aro4Delta, restoring the specific activity of the enzyme (a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase) to wild-type levels. DhARO4 transcript expression under high salinity was stimulated at the beginning of the exponential growth phase. As the DhARO4 promoter region presents putative GCRE and CRE sequences, its expression was evaluated under conditions of NaCl stress and amino acid starvation, showing similar expression levels for either condition. The combined effect of both stressors resulted in a further increase in transcript levels over the singly added stressors, indicating independent stimulatory events. Our results support the hypothesis that high salinity and amino acid availability are physiologically interconnected.