The Spoilage Yeast Zygosaccharomyces bailii Forms Mitotic Spores: a Screening Method for Haploidization

Zygosaccharomyces bailii ISA 1307 and the type strain of thisspoilage yeast show a diploid DNA content. Together with a ratherpeculiar life cycle in which mitotic but no meiotic spores appearto be formed, the diploid DNA content explains the observeddifficulties in obtaining auxotrophic mutants. Mitotic chromosomeloss induced by benomyl and selection on canavanine media resultedin three haploid strains of Z. bailii. This new set of Z. bailiistrains allows the easy isolation of recessive mutants and issuitable for further molecular genetic studies.

INTRODUCTION

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Introduction

The genus Zygosaccharomyces is associated with a notorious resistanceto stress environments (12, 22, 27). It includes different species,such as Z. bailii, Z. bisporus, and Z. rouxii, which have beenfrequently implicated in the spoilage of food and beverages(2). A screen for yeasts highly resistant to weak carboxylicacids involving a wide range of genera revealed that the strainISA 1307 of Z. bailii, originally isolated from continuous productionof sparkling wine (30), displayed an outstanding capacity tosurvive in such environments. Whereas different physiologicaland biochemical mechanisms have been identified to explain theresistance of Z. bailii to acidic environments (26), the molecularbasis of such behavior is unknown. Molecular tools for the studyof this species have begun to appear recently. These includegenomic libraries and genes suitable for complementation ofauxotrophic markers (17, 20). However, previous attempts toisolate auxotrophic mutants of Z. bailii failed, possibly dueto a ploidy higher than haploidy (16). We therefore set outto construct a set of Z. bailii strains suitable for mutationalanalysis, thus widening the possibilities for further moleculargenetic studies of this organism.

The strains ISA 1307 and IGC 5167 (the type strain) of the yeastZ. bailii were used. The strain W303 1A of Saccharomyces cerevisiae(MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1) was used asa reference for DNA quantification. Escherichia coli XL1-Bluewas used as the bacterial host for plasmids (5). Yeast strainswere grown in yeast extract-peptone-dextrose (21) or in syntheticmedium (yeast nitrogen base [YNB]; Difco) with the necessarybases and amino acids (23). L-Canavanine (60 µg/ml; Sigma),Geneticin (G418 sulfate, 20 µg/ml; Invitrogen), 5-fluorooroticacid (0.75 mg/ml; Sigma), or cycloheximide (0.1 and 0.4 mg/liter;Sigma) was used. E. coli strains were grown in Luria-Bertanimedium at 37°C (21). Standard yeast genetic procedures wereused for sporulation of Z. bailii and dissection of asci (3,24), for DNA manipulation (20, 21), and for preparation of competentcells (10, 13). Mutations were induced by exposing yeast cellsto UV light (HNS 30-W OFR; Osram) at a distance of 60 cm. Formitotic chromosome loss, cells were inoculated at 10⁷ cells/mlin YNB containing benomyl (30 or 100 µg/ml) at 23°C.After 20 h, cells were harvested from the culture medium, washedto remove the benomyl precipitate, and inoculated at 26°Cin YNB containing L-canavanine (60 µg/ml). For fluorescencemicroscopy analysis, cells from a mid-log growth phase wereused (21). Colocalization of DsRed-NLS (red fluorescent proteintagged into the nuclei), yEGFP3-NLS (green fluorescent proteintagged into the nuclei), and DNA was performed as describedelsewhere (21). Sporulated cells of Z. bailii were collectedfrom solid media and stained with calcofluor white (18) or 4',6'-diamidino-2-phenylidole(DAPI; Merck). For flow cytometry experiments (Partec PAS flowcytometer, equipped with an argon-ion laser emitting a 488-nmbeam at 15 mW), the DNA in whole cells was stained with SYBRGreen I (Molecular Probes) (9). The cell cycle histograms presentedwere obtained by offline analysis of list mode files with WinMDI2.5 software.

Z. bailii is diploid but forms tetrads with mitotic spores.

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Z. bailii is diploid...

Several groups have tried to select for auxotrophic strainsof Z. bailii without success (16, 17). Initial attempts to obtainUra^-, Leu^-, Trp^-, Ade^-, or His^- auxotrophic mutants of Z. bailiiISA 1307 by exposure to UV irradiation (Fig. 1) have failed.At a survival rate of 10%, around 8,000 colonies were analyzedand all were still prototrophic. Similarly, a positive selectionfor auxotrophic mutants (4, 29) using 5-fluoroorotic acid or5-fluoroanthranilic acid was unsuccessful. All 5-fluorooroticacid-resistant colonies (90 colonies per 10⁹ cells) were stillUra⁺. Identical results were obtained for the type strain ofZ. bailii. All together, these results pointed to a ploidy ofstrain ISA 1307 higher than haploidy. This idea was reinforcedby the higher UV resistance of the Z. bailii strains than thatof a haploid S. cerevisiae strain (Fig. 1). Most convincingly,the disruption of the ACS2 gene of Z. bailii (ZbACS2) (accessionnumber AJ314837, encoding the acetyl coenzyme A synthetase)in Z. bailii ISA 1307 with the APT1 gene, which confers resistanceto the aminoglycoside G418 (Geneticin) (11), resulted in thestrain Z. bailii ISA 1307-14, which has a heterozygous ZbACS2/Zbacs2::APT1background (Fig. 2). Furthermore, all 80 viable spores isolatedfrom complete tetrads of this strain were G418 resistant. Moreover,all 16 spores from four complete tetrads were heterozygous forthe ZbACS2/Zbacs2::APT1 alleles (F. Rodrigues, unpublished data).These observations and similar ones described by Mollapour andPiper (17) suggested that Z. bailii forms mitotic rather thanmeiotic spores. By using calcofluor white staining (18), weshowed the presence of several bud scars in each cell involvedin the conjugation process, indicating that both cells had alreadybudded several times (Fig. 3, section I). This result pointsto the fact that two different cells are involved in the formationof the ascus-like structure; otherwise, if conjugation betweenmother and daughter cells occurs, one could expect the presenceof several scars in just one of the cells. To further explainthe formation of mitotic spores, the fusion of the two nucleiduring this process was tested by crossing cells with differentlabeled nuclei (19). The DsRed-NLS and yEGFP3-NLS genes werecloned in vectors that self-replicate in Z. bailii with G418as the dominant resistance marker. Z. bailii strains expressingeach one of the fluorescent proteins were obtained, showingthat the proteins were tagged into nuclei (result not shown).Unfortunately, cells expressing those fluorescent proteins lostfluorescence during the induction of sporulation, even whenthe strong S. cerevisiae ADH1 promoter was used. Changes ingene expression also occurred during sporulation in S. cerevisiae(7, 15); however, because no known promoter sequence of Z. bailiiwas up regulated under this condition, the utilization of thistechnology after the conjugation process was hampered. To furtherbroaden the scope of these results, we tested whether nuclearfusion would occur during sporulation by DAPI staining of DNA(Fig. 3, section II). From 500 conjugated but not yet sporulatedcells, only one showed fused nuclei. The low frequency of nuclearfusion seems to indicate that such an event does not occur duringconjugation. In addition, we observed that sporulated cellsof Z. bailii ISA 1307 showed only one nucleus per ascospore(Fig. 3, section III). In conclusion, the absence of nuclearfusion during sporulation in Z. bailii seems to cause mitoticspore formation.

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FIG. 1. Survival curves for Z. bailii ISA 1307 (filled circles), Z. bailii H15 (one of the isolated haploid mutants [open circles]), and S. cerevisiae W303 1A (filled squares) after irradiation with UV light.

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FIG. 2. Disruption of ZbACS2 (accession number AJ314837) by APT1. (A) Schematic representation of the APT1 expression cassette for disruption experiments. (B) Southern blot of the Z. bailii ISA 1307 wild-type strain (lanes 1 and 3) and mutant 14, which is resistant to Geneticin (Z. bailii ISA 1307-14) (lanes 2 and 4). The genomic DNA isolated was digested with the restriction enzyme HindIII (lanes 1 and 2) or PstI-XhoI (lanes 3 and 4).

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FIG. 3. Microscopy of stained Z. bailii cells. (A) Bright field; (B) blue fluorescence; (C) overlap of bright field and blue fluorescence. (Section I) Conjugated cells stained with calcofluor white; (section II) conjugated but not sporulated cells stained with DAPI; (section III) sporulated cells stained with DAPI. The fluorescence of calcofluor white and DAPI is blue.

Haploid mutants of Z. bailii.

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Haploid mutants of z....

Since sporulation of Z. bailii ISA 1307 did not lead to haploidcells, haploidization was induced by growth on synthetic mediacontaining benomyl. This drug induces mitotic chromosome lossat high frequencies (31). The CAN1 gene, encoding an argininepermease (1, 8, 25), was employed as a recessive resistancemarker for the selection of haploid mutants. Canavanine, a toxicarginine analog to which can1 mutants are resistant, was usedto select for spontaneous mutants. After 4 days of incubationat 30°C, colonies were found at a frequency of approximatelyone mutant per 1.4 x 10⁸ cells plated. Fifty putative haploidmutants were recovered and further characterized with respectto DNA content, ability to sporulate, and production of recessivemutations. Cell DNA content was estimated by flow cytometrywith the DNA fluorescent probe SYBR Green I (6, 9). Flow cytometryof stained exponentially growing cells of S. cerevisiae W3031A, Z. bailii ISA 1307, Z. bailii IGC 5167, and of one isolatedmutant (H15) evidenced the presence of G₀/G₁ and G₂/M peaks(Fig. 4) indicative of different DNA contents. Cell cycle analysisrevealed the mean fluorescence intensities (MFI) of the G₀/G₁and G₂/M peaks (Table 1) with a half-peak coefficient of variation(HPCV) lower than 7%, an indication of the high precision ofthe measurements. The DNA index (DNA In) was defined as theratio between the fluorescence intensity of the G₀/G₁ peak ofthe strain to be tested and that of a reference strain. Twodifferent DNA Ins were calculated, one in relation to S. cerevisiaeW303 1A (DNA In-1) and the other in relation to Z. bailii ISA1307 (DNA In-2) (Table 1). The values of DNA In-1 for the Z.bailii type strain and ISA 1307 indicate that they have, respectively,1.4 and 2.1 times as much DNA as S. cerevisiae W303 1A. TheDNA In-2 revealed the DNA contents of strains H15, H50, andH51 to be about half that of the parental strain (Table 1).All the other canavanine-resistant mutants isolated had DNAIn-2 values ranging from 0.6 to 1 and were discarded due totheir purported aneuploid status. Considering the genome sizesof the type strain of Z. bailii (7.7 Mb) (28) and of S. cerevisiae(13.1 Mb) together with the MFI of the G₀/G₁ peak (Table 1),the type strain of Z. bailii is, most likely, diploid. Pulsed-fieldgel electrophoresis showed that Z. bailii ISA 1307 has at leastthree more chromosomes than the type strain, two of higher massesand one of lower molecular mass (data not shown), which mightexplain the apparent higher DNA content of this strain thanthat of the type strain. All together, these results also pointto a diploid DNA content of Z. bailii ISA 1307 and consequentlyto a haploid or near-haploid state of strains H15, H50, andH51. We next tested whether these three strains could producerecessive mutants. Indeed, a high number of spontaneous cycloheximide-resistantmutants was selected for from those strains (14, 25). The strainsH15, H50, and H51 were also tested for the ability to sporulate.Whereas H15 was able to sporulate, spores were not found inthe two other strains. Therefore, strains H50 and H51 were eithersterile or heterothallic and were of the same mating type, sinceno sporulation was detected after both strains were mixed. Finally,mutations were induced in strain H15 by UV radiation (Fig. 1).As expected, because of its DNA content, this mutant was moresensitive to radiation than the wild-type strain (ISA 1307),and an auxotrophic mutant for histidine was easily isolated.Thus, this new set of Z. bailii strains is suitable for furthermolecular genetic studies.

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FIG. 4. Green fluorescence histograms of S. cerevisiae W303 1A (A), Z. bailii ISA 1307 (B), Z. bailii H15 (C), and a mixture of Z. bailii ISA 1307 and Z. bailii H15 (D) after SYBR Green I staining.

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TABLE 1. Comparison of yeast genome sizes as determined by flow cytometry^a

The method described herein could also be very useful for othernonconventional yeasts with a diploid DNA content, as is thecase for some pathogenic yeasts. While we feel that the possibilityof readily isolating recessive mutants is an important stepforward, we realize that this is only a first step. The isolationof more haploid Z. bailii mutants will show us whether matingand meiosis will also become available as tools in this organism.

ACKNOWLEDGMENTS

We thank Filipe Sansonetty for his helpful discussion on cytometryresults and Anne-Marie Zeeman for her technical assistance onthe karyotyping of Z. bailii strains.

Fernando Rodrigues was the recipient of a fellowship from PRAXISXXI, and the study was supported by a research grant from theFundação para a Ciência e Tecnologia, Lisbon,Portugal (contract PRAXIS XXI P/AGR/11135/98).

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Biologia, Universidade do Minho, 4710-057 Braga, Portugal. Phone: 351 253 604314. Fax: 351 253 678980. E-mail: mcortereal{at}bio.uminho.pt.

REFERENCES

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