INTRODUCTION {#sec1}
============

*Zygosaccharomyces bailii* is found among the spoilage yeasts that frequently affect acidic foods and beverages, particularly mayonnaise, salad dressings, soft drinks, fruit concentrates and dairy products (reviewed in Stratford [@bib63]; Sá-Correia *et al*. [@bib55]). This yeast species ability to cause spoilage results from its remarkable capacity to tolerate stress induced by weak acids widely used as fungistatic preservatives, such as acetic, benzoic and sorbic acids (Ferreira, Loureiro-Dias and Loureiro [@bib15]; Stratford *et al.*[@bib64]). In fact, *Z. bailii* may proliferate in the presence of weak acid concentrations above those legally permitted in food products (Stratford [@bib63]). Moreover, *Z. bailii* is able to tolerate relatively high ethanol concentrations (Kalathenos, Sutherland and Roberts [@bib25]) and high temperatures (Martorell *et al.*[@bib33]), and to vigorously ferment sugars (Thomas and Davenport [@bib72]), being considered one of the most significant threats in wine industry (Loureiro and Malfeito-Ferreira [@bib30]). Like other members of the *Zygosaccharomyce*s genus, *Z. bailii* is fructophilic, i.e. prefers fructose over glucose when both sugars are present in the growth medium (Sousa-Dias *et al.*[@bib61]; Pina *et al.*[@bib41]). Therefore, acidic food and beverages that contain fructose are at particular risk of spoilage, given that *Z. bailii* specific growth rates and ethanol production are higher during growth in the presence of fructose compared with glucose. Although *Z. bailii* is unable to directly metabolize sucrose, acidic products containing this sugar are also susceptible to spoilage by this yeast species given that, at low pH, sucrose can be hydrolyzed in fructose and glucose (Pitt and Hocking [@bib44]). These traits of *Z. bailii* physiology result in annual losses of millions of dollars (Pitt and Hocking [@bib44]) making this species a serious concern in food industry. Regardless of its activity as spoilage yeast, *Z. bailii* has also gained attention for its potential in biotechnological processes, being proposed as a new cell factory for the production of organic acids and heterologous proteins (Branduardi *et al.*[@bib5]; Dato *et al.*[@bib10]).

Although there are plasmid vectors available for *Z. bailii* genetic manipulation (Branduardi, Dato and Porro [@bib4]), the genetic engineering of this species has been hindered by the lack of stable haploid strains (Mollapour and Piper [@bib36]; Rodrigues *et al.*[@bib50]) and, to date, only one auxotrophic mutant was constructed (Dato *et al.*[@bib10]). However, the parental strain of the derived mutant, previously considered as *Z. bailii*, was taxonomically reallocated to *Z. parabailii* (Suh *et al.*[@bib65]). Moreover, only recently it was possible to have access to the genome sequence of a *Z. bailii* strain, the reference strain CLIB 213^T^ (Galeote *et al.*[@bib17]) and to *Z. bailii*-derived hybrid strain ISA1307 (Mira *et al.*[@bib34]). This hybrid strain has been used in several studies aiming at understanding *Z. bailii* physiology, in particular the mechanisms underlying acetic acid tolerance and toxicity (Sousa *et al.*[@bib60]; Rodrigues *et al.*[@bib52]; Guerreiro, Mira and Sá-Correia [@bib18]; Palma *et al.*[@bib40]), but following its genome sequencing and annotation ISA1307 emerged as an interspecies hybrid between *Z. bailii* and a closely related species (Mira *et al.*[@bib34]). On the other hand, *Z. bailii* CLIB 213^T^ is extremely difficult to manipulate in the laboratory mainly due to its strong aggregation phenotype and, for this reason, strains CBS 685 (NCYC 563) and NCYC 1766 were suggested as better representatives of the species (James and Stratford [@bib24]). More recently, our laboratory has been working with *Z. bailii* IST302, which was found to be more amenable to genetic and physiological manipulations than strain CLIB 213^T^ (Palma *et al.*[@bib40], [@bib39]). Therefore, the sequencing of its genome sequence was considered the next step.

In this work, we provide molecular evidences supporting the taxonomic identification of strain IST302 as *Z. bailii*, as well as its genome sequence and annotation and comparative analysis with CLIB 213^T^ and other relevant yeast species. This study also envisaged the confirmation of the haploid nature of the genome, the characterization of relevant tolerance phenotypes and the search for potential molecular targets involved in *Z. bailii* remarkable weak acid tolerance and the aggregation phenotype. Collectively, the information gathered in this study points *Z. bailii* IST302 strain as highly attractive for the functional analysis of non-essential *Z. bailii* genes and the elucidation of mechanisms underlying its high tolerance to acetic acid and other weak acid food preservatives or to the investigation of *Z. bailii* potential as cell factory.

MATERIALS AND METHODS {#sec2}
=====================

Strains and growth medium {#sec2-1}
-------------------------

The prototrophic strains *Zygosaccharomyces bailii* IST302 (Palma *et al.*[@bib40]), the neotype strain *Z. bailii* ATCC58445^T^ (= CLIB 213^T^) (Galeote *et al.*[@bib17]) and strain ISA1307, a *Z. bailii*-derived interspecies hybrid strain, were used in this work. *Saccharomyces cerevisiae* BY4741 (genotype *MAT*a; *his3*Δ*1*; *leu2*Δ*0*; *lys2*Δ*0*; *ura3*Δ*0*) and BY4743 (*MAT*a/α *his3*Δ*1/his3*Δ*1 leu2*Δ*0/leu2*Δ*0 LYS2/lys2*Δ*0 met15*Δ*0/MET15 ura3*Δ*0/ura3*Δ*0)* were acquired from the EUROSCARF collection. Yeast strains were maintained in solid rich YPD growth medium that contains, per liter, 2 % glucose (Merck, Kenilworth, New Jersey), 1 % yeast extract (Difco Laboratories, Detroit, Michigan) and 2% peptone (Difco Laboratories, Detroit, Michigan) and 2% agar. For susceptibility assays, yeast strains ISA1307, IST302 and BY4741 were cultured in MM4 liquid medium that contains, per litre: 1.7 g yeast nitrogen base without amino acids or (NH~4~)~2~SO~4~ (Difco Laboratories, Detroit, Michigan), 20 g glucose (Merck, Kenilworth, New Jersey), 2.65 g (NH~4~)~2~SO~4~ (Merck), 20 mg methionine, 20 mg histidine, 60 mg leucine and 20 mg uracil (all from Sigma-Aldrich, St. Louis, Missouri).

Taxonomic identification of strain IST302 {#sec2-2}
-----------------------------------------

The nucleotide sequences of the genes from strain IST302 encoding the RNA polymerase II largest subunit Rpb1 and the RNA polymerase II second-largest subunit Rpb2, previously considered relevant for the identification of *Z. bailii* species among closely related species such as *Z. parabailii* and *Z. pseudobailii* (Suh *et al.*[@bib65]), were compared with the nucleotide sequences of *RPB1* and *RPB2* from several *Z. bailii*, *Z. parabailii* and *Z. pseudobailii* strains as described previously (Suh *et al.*[@bib65]). The nucleotide sequences of *RPB1* and *RPB2* genes from strain IST302 were obtained by whole-genome sequencing, as described hereafter.

Cell cycle analysis and quantification of *Zygosaccharomyces bailii* IST302 total genomic DNA by flow cytometry {#sec2-3}
---------------------------------------------------------------------------------------------------------------

*Zygosaccharomyces bailii* IST302 cells were batch cultured in YPD growth medium, at 30°C, until mid-exponential phase (OD~600nm~ = 0.6±0.1). A total of 10^7^ cells were harvested by centrifugation, washed twice with distilled water and fixed at least one night in 1 mL of 70 % ethanol (vol/vol). The remaining cell culture was induced to arrest at the G1 phase with 8-hydroxyquinoline (Sigma-Aldrich, St. Louis, Missouri) at a final concentration of 100 μg/mL for 24 h or at the G2 phase with nocodazole (Sigma-Aldrich, St. Louis, Missouri) at a final concentration of 5 μg/mL for 3 h. Arrested cells were harvested, washed and fixed with ethanol, as previously described. Quantification of total genomic DNA from *Z. bailii* IST302 by flow cytometry was performed using a SYBR Green I-based staining protocol, as described before (Fortuna *et al.*[@bib16]). Cells fixed with ethanol were collected by centrifugation, washed with 50 mM of sodium citrate buffer (pH 7.5) and resuspended in 750 μL of this same buffer supplemented with 250 μL of RNAse A (1 mg/mL) (Sigma-Aldrich, St. Louis, Missouri). After 1 h of incubation at 50°C, 50 μL of proteinase K (20 mg/mL) (NZYtech, Lisbon, Portugal) were added to the cell suspension and the mixture was incubated at 50°C for another hour. Cells were subsequently stained overnight at 4°C using SYBR^®^ Green I at a final concentration of 500-fold dilution of the commercial SYBR^®^ Green I (Life Technologies, Carlsbad, California). Triton X-100 was added to the stained samples at a final concentration of 0.25% (v/v). Samples were vortexed, sonicated in a Branson Sonifier 250 (3-4 pulses with at output power of 3 and 30% duty cycle) and analyzed in a FACScalibur (Becton Dickson, Franklin Lakes, New Jersey) flow cytometer. A minimum of 50000 cells per sample were acquired at low flow rate using CellQuest software (Becton Dickson, Franklin Lakes, New Jersey). Analysis of the acquired data was performed using the FlowJo® v10.0.8 software. The fluorescence intensities of the cell cycle peak G0/G1 for *S. cerevisiae* BY4741 and BY4743 were used to build a calibration curve in order to estimate the genome size of *Z. bailii* IST302 strain. Fluorescence intensities for the hybrid strain ISA1307 were used for comparison purposes.

Karyotyping of *Zygosaccharomyces bailii* IST302 strain {#sec2-4}
-------------------------------------------------------

DNA for pulsed field gel electrophoresis (PFGE) was prepared in plugs as previously described (Maringele and Lydall [@bib32]). Strains IST302 and CLIB 213^T^ were cultivated overnight at 30°C at 250 rpm in YPD growth medium. Yeast cells were harvested by centrifugation in order to obtain a cell pellet of ∼50 μL volume. Cell pellets were washed twice with 0.05 M EDTA, pH 8.0 and resuspended in 100 μL of 0.05 M EDTA, pH 8.0. Plugs were formed by mixing the suspension of cells with a total of 50 μL of a Zymolyase solution \[46 μL SCE buffer (1 M sorbitol, 0.1 M sodium citrate, and 60 mM EDTA), 1.5 μL zymolyase (NZYtech, Lisbon, Portugal) and 2.5 μL β-mercaptoethanol\] was added to each sample followed by the addition of 300 μL of low-melting point agarose solution (1% low melting point agarose in 10 mL EDTA 0,125 mM, pH 7.0). The mixture was pipetted into wells from the plug molds and placed at 4°C during 30 min. The plugs were then carefully removed and then incubated overnight at 37°C, 50 rpm, in ETB buffer (EDTA 0.05 M, pH 8.0, Tris-HCl 0.1 M, pH 8.0 and 5 % (vol/vol) β-Mercaptoethanol). After this incubation step, plugs were washed three times in TE buffer (10 mM Tris, pH 8.0 and 1 mM EDTA, pH 8.0) and incubated overnight at 37°C in a solution containing 0.05 M EDTA, 1 mg/mL of proteinase K (NZYtech, Lisbon, Portugal), 1 mg/mL RNAse (Sigma-Aldrich, St. Louis, Missouri) and 1 % sodium-N-lauryl sarcosinate. The plugs were washed with EDTA 50 mM at 37°C, 100 rpm, during 15 mins and incubated at room temperature for 1 h in TE buffer, pH 8.0. PFGE was performed in a CHEF- Pharmacia LKB Gene Navigator system. PFGE gels were run in 0.5 % Tris borate--EDTA buffer at 13°C with a voltage of 3 V/cm and switch times of 600 s for 48 h and 300 s for 96 h in a 1 % pulsed-field gel agarose (NZYtech, Lisbon, Portugal).

Genome sequencing, assembly and annotation {#sec2-5}
------------------------------------------

*Zygosaccharomyces bailii* IST302 genome sequencing and assembly was performed as described previously (Mira *et al.*[@bib34]) using a whole-genome shotgun approach that explored paired-end Illumina sequencing (Illumina Hiseq 2000 system, CD Genomics, New York, USA). Short reads were assembled using SOAPdenovo (<http://soap.genomics.org.cn>) (Li *et al.*[@bib27]). The obtained scaffolds were sequentially ordered based on their level of synteny to the genomes of *Z. rouxii* CBS 732^T^, *S. cerevisiae* S288c, *Z. bailii*-derived hybrid strain ISA1307 and *Z. bailii* CLIB 213^T^. To predict genes on the scaffolds, two ab initio gene predictor algorithms were used: GeneMark-S and GenMark-ES version 2.3 (Ter-Hovhannisyan *et al.*[@bib71]). Gene models differently predicted by the algorithms were manually curated based on the structure obtained for *Z. rouxii* CBS 732^T^, *S. cerevisiae* S288c, *Z. bailii*-derived hybrid strain ISA1307 and *Z. bailii* CLIB 213^T^ homologs. The genomes were analyzed using the PEDANT system (Walter *et al.*[@bib74]) to allow comparative feature analysis. To avoid misleading ortholog information based on similarity and bidirectional best hits, QuartetS (Yu *et al*. [@bib72a]) was applied to retrieve a reliable ortholog matrix, which was used for comparative analysis of the genes encoding multidrug/multixenobiotic resistance (MDR/MXR) transporters, of the genes involved in life cycle and meiosis and of the genes involved in cellular aggregation in *Z. bailii* IST302, *Z. bailii* CLIB 213^T^, *Z. rouxii* CBS732^T^ and *S. cerevisiae* S288c. The identification of the centromeres was performed manually based on previously identified point centromere sequences and on the collinear conservation of flanking genes of hemiascomycetous yeasts, in particular of *Z. rouxii* (Pribylova *et al.*[@bib46]; Souciet *et al.*[@bib59]).

The genome sequence and annotation of *Z. bailii* IST302 has been deposited in ENA (<http://www.ebi.ac.uk/ena/data/view/FUGC01000001-FUGC01000105>).

Susceptibility assays {#sec2-6}
---------------------

Susceptibility of *Z. bailii* IST302, ISA1307 hybrid strain and *S. cerevisiae* BY4741 to several growth inhibitory compounds was assessed by spot assays. Yeast cells were grown in MM4 medium at the appropriate pH until exponential phase (OD~600nm~ of 0.5 ± 0.05) and then reinoculated at an OD~600nm~ of 0.05, in 50 mL of fresh medium. When the cultures reached an OD~600nm~ of 0.5 ± 0.05, cells were resuspended in sterile water to obtain suspensions with ∼5 × 10^5^ cell/mL. These cell suspensions and three subsequent dilutions (1:5; 1:10; 1:20) were applied as 4 μL spots on MM4 plates supplemented with adequate concentrations of acetic, benzoic, formic, lactic, propionic or sorbic acids; of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA) or alachlor; and of the fungicides itraconazole, fluconazole, miconazole, tioconazole, clotrimazole or mancozeb. Agar plates were incubated at 30°C for 3 days. Due to the different pKa of the acids, yeast susceptibility to acetic, benzoic, sorbic and propionic acids was tested in MM4 medium at pH 4.0, whereas susceptibility to formic and lactic acids was tested in MM4 at pH 3.5.

Sporulation of *Zygosaccharomyces bailii* IST302 {#sec2-7}
------------------------------------------------

*Zygosaccharomyces bailii* IST302 cells were grown in a presporulation medium (0.8% yeast extract, 0.3% peptone, 10% glucose and 2% agar) during 2 days before being transferred to sporulation media (0.3% malt extract, 0.5% peptone and 2% agar). The formation of spores was observed on a Zeiss ® Axioplan microscope (×1000 magnification) after 2 and 4 days of incubation at 30°C.

Microscopic observation of the morphology of yeast colonies and cells {#sec2-8}
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Yeast cells were plated in YPD with a targeted density of 20--50 CFU/plate. Colonies were observed and photographed in a Carl Zeiss Stemi 2000-C stereomicroscope after 4--5 days of growth at 30°C.

Cells of *Z. bailii* IST302, *Z. bailii* CLIB 213^T^ and of ISA1307 hybrid strain were cultured in YPD medium for 24 h and used to inoculate a fresh YPD medium with an initial OD~600nm~ of 0.05±0.01. After 24 h of growth, cells were harvested in 1.5 mL tubes by centrifugation at 8000 rpm and suspended in 0.05 M EDTA (pH 8.0), followed by vigorous agitation. These cells were observed on a Zeiss ® Axioplan microscope (×400 magnification). Cells suspended in 0.05 M EDTA (pH 8.0) were subsequently washed twice with sterile water, suspended in 0.01 M CaCl~2~, vigorously mixed and finally observed under the microscope.

RESULTS AND DISCUSSION {#sec3}
======================

Taxonomic identification of strain IST302 {#sec3-1}
-----------------------------------------

The molecular differences registered among *Zygosaccharomyces bailii* and closely related species (Suh *et al.*[@bib65]), together with the genomic complexity of the hybrid strain ISA1307 (Mira *et al.*[@bib34]) that was first considered as *Z. bailii*, support the notion of the remarkable genomic diversity among *Z. bailii* and *Z. bailii* closely related species. This fact recommended the careful examination of the taxonomic position and genomic characterization of IST302 strain. The preliminary taxonomic identification of this strain as *Z. bailii* was based on the comparison of the large subunit 26S ribosomal DNA partial sequence with other DNA sequences from *Z. bailii* strains using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) (Palma *et al.*[@bib40]). However, considering the reallocation of yeast strains formerly identified as *Z. bailii* to the new species *Z. parabailii* and *Z. pseudobailii* (Suh *et al.*[@bib65]), we have also compared the partial sequences of *RBP1* and *RBP2* genes from strain IST302 with their homologs from several strains of the three species, as recommended by Suh *et al*. ([@bib65]). Results show that nucleotide sequence variation percentage is low when *RBP1* and *RBP2* nucleotide sequences from strain IST302 are compared with the corresponding sequences from *Z. bailii* strains (0.1%--0.7%), whereas significant sequence variation is observed compared with *Z. parabailii* (3.8%--4.1%) and *Z. pseudobailii* (5.8%--6.8%) strains (Fig. [1](#fig1){ref-type="fig"}). Collectively, these results confirm IST302 strain as *Z. bailii*.

![Nucleotide variation in *RBP1* and *RBP2* genes from strain IST302 when compared with the partial sequence of *RBP1* and *RBP2* from *Z. bailii*, *Z. parabailii* and *Z. pseudobailii* strains. Nucleotide variations were calculated using EMBOSS Water local alignment tool from EMBL-EBI packages. Values are presented as percentage of nucleotide variations and are the mean of results obtained by comparing *RBP1* and *RBP2* sequences from strain IST302 (ZBIST_2664 and ZBIST_4878, respectively) with their homologs from six strains of *Z. bailii*, seven strains of *Z. parabailii* and two strains of *Z. pseudobailii* according to Suh *et al*. ([@bib65]).](fox025fig1){#fig1}

Tolerance of *Zygosaccharomyces bailii* IST302 to weak acids, drugs and pesticides {#sec3-2}
----------------------------------------------------------------------------------

The tolerance of *Z. bailii* IST302 to several compounds was assessed by spot assays (Fig. [2](#fig2){ref-type="fig"}). Compared with *Saccharomyces cerevisiae* BY4741*, Z. bailii* IST302 is much more tolerant to weak acids (acetic, benzoic, sorbic, propionic and formic acids), to the lipophilic weak acid herbicides 2,4-D (2,4-D-dichlorophenoxyacetic acid) and MCPA (methyl-chlorophenoxyacetic acid) and also to the herbicide alachlor, but not to the antifungals tested (Fig. [2A and B](#fig2){ref-type="fig"}). In general, *Z. bailii* IST302 is slightly more tolerant to weak acids than the hybrid strain ISA1307. Interestingly, the hybrid strain ISA1307 is significantly more tolerant to all the antifungal drugs tested than *Z. bailii* IST302 and *S. cerevisiae* BY4741 (Fig. [2C](#fig2){ref-type="fig"}). The standardization of the number of viable/total cells was not possible for *Z. bailii* CLIB 213^T^ due to the difficulty to calculate viable cell concentration or culture optical density. For this reason, only the susceptibility of *Z. bailii* IST302, the hybrid strain ISA1307 and *S. cerevisiae* BY4741 to several compounds could be compared under equivalent experimental conditions.

![*Z*ygosaccharomyces *bailii* IST302 is more tolerant to weak acid food preservatives and herbicides, but not to antifungals, when compared with *S. cerevisiae* BY4741. Growth of *Z. bailii* IST302, *S. cerevisiae* BY4741 and the hybrid strain ISA1307 was compared by spot assays in MM4 medium supplemented, or not, with the appropriate concentrations of (**A**) weak organic acids, (**B**) herbicides and (**C**) antifungal drugs. Three control plates are shown, with and without the solvents used to dissolve the different drugs when necessary. Cell suspensions with ∼5 × 10^5^ cells/mL (lane a) and subsequent dilutions of 1:5, 1:10 and 1:20 (lanes b, c and d, respectively) were spotted onto the surface of MM4 solid medium. The images shown were taken after 3 days of incubation at 30°C and are representative of at least three independent experiments.](fox025fig2){#fig2}

Estimation of total DNA content and karyotyping of *Zygosaccharomyces bailii* IST302 {#sec3-3}
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The chromosomal profiles of *Z. bailii* IST302 and of the reference strain *Z. bailii* CLIB 213^T^ were compared by PFGE (Fig. [3](#fig3){ref-type="fig"}). Karyotyping showed differences in the number and size of the chromosomes between *Z. bailii* IST302 and CLIB 213^T^ strains. Following the separation of chromosomes by PFGE, strain IST302 originated a total of seven DNA bands, whereas only six DNA bands are observed for CLIB 213^T^. Based on the molecular size of chromosomes from the marker species *Hansenula wingei*, it was possible to estimate the total DNA content of *Z. bailii* IST302 and CLIB 213^T^ as 11.65 and 9.5 Mb, respectively. Given that the DNA content calculated from CLIB 213^T^ is lower than the DNA sequenced (Galeote *et al.*[@bib17]), it is possible that two comigrating chromosomes could have not been separated using the PFGE conditions tested. In fact, the chromosomal DNA band of 1.8 Mb is more intense than expected for a single band and possibly represents two chromosomes of the same size.

![Karyotype profiles of *Z. bailii* IST302 and CLIB 213^T^ strains. Separation of the chromosomes was performed by PFGE as described in Materials and Methods section. The size of *Z. bailii* IST302 and CLIB 213^T^ chromosomes was estimated from a comparison with the size of the chromosomes from *Hansenula wingei* that were used as the molecular size standards (M).](fox025fig3){#fig3}

Flow cytometry was also used to estimate the genome size of Z*. bailii* IST302, as well as its ploidy. *Saccharomyces cerevisiae* BY4741 was used as the haploid reference strain and *S. cerevisiae* BY4743 as the diploid reference. The hybrid strain ISA1307, whose genome size and ploidy were recently estimated (Mira *et al.*[@bib34]), was also included in this analysis for comparison purposes. *Zygosaccharomyces bailii* CLIB 213^T^ was excluded from this analysis due to its strong cellular aggregation phenotype. As anticipated, the fluorescent light intensity of G0/G1 or G2/M phase cells from the reference haploid strain *S. cerevisiae* BY4741 is half the fluorescent light intensity of the diploid BY4743 strain, since strain BY4741 possesses half the DNA content of strain BY4743 (Fig. [4A and B](#fig4){ref-type="fig"}). Flow cytometry data from strain ISA1307 (Fig. [4C](#fig4){ref-type="fig"}) shows two fluorescence peaks (G0/G1 and G2/M) with a DNA content similar to one of the diploid *S. cerevisiae* BY4743 (Fig. [4B](#fig4){ref-type="fig"}). Interestingly, strain IST302 (Fig. [4D](#fig4){ref-type="fig"}) shows two fluorescence peaks (G0/G1 and G2/M) with a DNA content similar to the one observed for *S. cerevisiae* BY4741 haploid strain (Fig. [4A](#fig4){ref-type="fig"}). Given the unexpected high proportion of *Z. bailii* IST302 cells in G2/M phase, it was decided to treat cells with 8-hydroxyquinoline and nocodazole in order to arrest cells in G0/G1 (Fig. [4E](#fig4){ref-type="fig"}) and in G2/M (Fig. [4F](#fig4){ref-type="fig"}) cell cycle phases, respectively, thus confirming the fluorescent peak corresponding to each phase. Results suggest that *Z. bailii* IST302 has a haploid DNA content (Fig. [4D](#fig4){ref-type="fig"}). Based on the fluorescent intensity of G0/G1 subpopulation from *S. cerevisiae* haploid and diploid strains, the size the genome of *Z. bailii* IST302 was estimated as having ∼11 Mb. The life cycle of the strain will be discussed further in this work.

![Cell cycle analysis histograms of *S. cerevisiae* BY4741 (haploid) (**A**) and BY4743 (diploid) (**B**), of the hybrid strain ISA1307 (**C**) and of *Z. bailii* IST302 (**D--F**). Fluorescent intensities of G0/G1 peaks of the cell cycle histograms were estimated by flow cytometry. The mean fluorescence values (FL1-**H**) obtained for *S. cerevisiae* haploid (A) and diploid (B) strains were used to build a calibration curve that was used to estimate the size of the genome of *Z. bailii* IST302 (D). *Zygosaccharomyces bailii* hybrid strain ISA1307 was used for comparison purposes (C). *Zygosaccharomyces bailii* IST302 cells were arrested in G0/G1 with 8-hydroxyquinoline (E) or in G2/M with nocodazole (F) in order to confirm the G0/G1 and G2/M peaks and determine the ploidy of the strain.](fox025fig4){#fig4}

Assembly and annotation of *Zygosaccharomyces bailii* IST302 genome sequence {#sec3-4}
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The genome sequence of *Z. bailii* IST302 was obtained by paired-end Illumina sequencing. A total of 13 million reads were acquired and assembled into 105 scaffolds, resulting in an overall sequence coverage of ×120. A summary of genome assembly statistics and its comparison with *Z. bailii* CLIB 213^T^ and the *Z. bailii*-derived interspecies hybrid ISA1307 is presented in Table [1](#tbl1){ref-type="table"}. The sum of all scaffolds size is 10 772 966 bp, which corresponds to 98% of the genome size estimated by flow cytometry and 93% of the genome size estimated by PFGE. The annotation of *Z. bailii* IST302 genome sequence was based on the combination of two ab initio gene predictor algorithms and the gene structure of *Z. rouxii* CBS 732^T^, *S. cerevisiae* S288c, *Z. bailii*-derived hybrid strain ISA1307 and *Z. bailii* CLIB 213^T^. A total of 5142 genes were predicted to be encoded in the genome of *Z. bailii* IST302 (Table [2](#tbl2){ref-type="table"}), whereas 5084 putative protein-coding genes were predicted to be encoded in the genome of *Z. bailii* CLIB 213^T^ (Galeote *et al.*[@bib17]). About 80% of the annotated genes are located in the first 25 scaffolds and 47% of the scaffolds contain zero or one gene annotated.

###### 

Genome assembly statistics of *Z. bailii* IST302.

                               IST302       ISA1307 (Mira *et al*. [@bib34])   CLIB 213^T^ (Galeote *et al*. [@bib17])
  ---------------------------- ------------ ---------------------------------- -----------------------------------------
  Total reads                  13 024 866   120 000 000                        50 868 918
  No. of scaffolds             105          154                                56
  Coverage                     ×120         ×600                               ×250
  N50 (bp)                     432084       232974                             932251
  Maximum contig length (bp)   1149457      806952                             1686157
  Minimum contig length (bp)   1051         2160                               
  Average contig length (bp)   102599       137280                             
  Assembly size (bp)           10772966     21141152                           10361356

###### 

Comparison of *Z. bailii* and *Z. bailii*-related strains genome features.

                                      No. of chromosomes   Ploidy   Genome   Average GC   Total no.
  ----------------------------------- -------------------- -------- -------- ------------ -----------
  *Z. bailii* IST302                  7                    ∼*n*     ∼11.0    42.2         5142
  *Z. bailii* CLIB 213^T^             6                    nd       ∼10.4    42.5         5084
  *Z. bailii* hybrid strain ISA1037   13                   ∼*2n*    22.0     42.4         9931

nd. Not determined

As expected, a high degree of conservation is observed between *Z. bailii* IST302 and CLIB 213^T^ strains. However, the comparison of these *Z. bailii* strains with the taxonomically related yeast species *Z. rouxii* reveals numerous chromosomal rearrangements (Fig. [5](#fig5){ref-type="fig"}).

![Multigenome alignment of genomic regions of *Z. bailii* IST302, *Z. bailii* CLIB 213^T^ and *Z. rouxii* CBS 732^T^. *Zygosaccharomyces bailii* IST302 is presented as the central reference strain to which the others are compared. Conserved synteny blocks are presented in shaded boxes. The image was obtained using the multigenome alignment Gbrowse_syn (McKay, Vergara and Stajich [@bib31]).](fox025fig5){#fig5}

A total of seven centromere-like sequences were identified in the genome sequence of IST302 (Supplementary file 1, Supporting Information), based on previously identified point centromere sequences and on the collinear conservation of flanking genes of hemiascomycetous yeasts, in particular of *Z. rouxii* (Pribylova *et al.*[@bib46]; Souciet *et al.*[@bib59]). This result is in agreement with the seven chromosomal bands identified during the karyotyping analysis (Fig. [3](#fig3){ref-type="fig"}). Interestingly, seven centromere-like sequences were also identified in the genome sequence of CLIB 213^T^ strain (Supplementary file 2, Supporting Information), thus reinforcing that the separation of the chromosomes by PFGE (Fig. [3](#fig3){ref-type="fig"}) was not complete for two of the chromosomes that possess highly similar sizes. It is also likely that, given the different sizes of both *Z. bailii* strains chromosomes and the conservation of collinearity, several genetic events might have occurred leading to different chromosome sizes. Strain-specific diversity of chromosome copy number and/or size has also been reported in other yeast species, such as *Z. rouxii* (Solieri *et al.*[@bib58]), *S. cerevisiae* (Zhu, Sherlock and Petrov [@bib77]), *Debaryomyces hansenii* (Jacques *et al.*[@bib23]), *Candida albicans* (Rustchenko-Bulgac [@bib53]), *C. glabrata* (Muller *et al.*[@bib37]) and *Dekkera bruxellensis* (Hellborg and Piskur [@bib19]).

The life cycle of *Zygosaccharomyces bailii* IST302 {#sec3-5}
---------------------------------------------------

The analysis of *Z. bailii* IST302 genes involved in life cycle and meiosis (Table [3](#tbl3){ref-type="table"} and Supplementary file 3, Supporting Information) revealed that this strain holds homologs of *S. cerevisiae* mating pheromone alpha- and a- factors, *MF(ALPHA)1* and *MFA1*, respectively, and a homolog of *S. cerevisiae MATALPHA1* that codes for a transcriptional co-activator of mating type specific genes. Neither *MATALPHA2* nor *MATA1/2* genes were detected in the genome sequence of this strain, suggesting that this haploid strain is of alpha mating type. Remarkably, although a homolog of the endonuclease encoding gene *HO*, responsible for mating type switching in *S. cerevisiae*, is present in *Z. bailii* IST302 genome, the silent cassettes HML/HMR were not identified in the genome sequence of this strain, neither in CLIB 213^T^ (Table [3](#tbl3){ref-type="table"}). In the context of evolution of hemiascomycete yeasts, the absence of silent cassettes is unexpected given that *Z. rouxii*, a close relative of *Z. bailii*, possesses those cassettes (Butler *et al.*[@bib7]; Fabre *et al.*[@bib13]; Watanabe, Uehara and Mogi [@bib75]). Although the hypothesis that HML/HMR cassettes were not sequenced cannot be discarded, the fact that they were not identified in this study nor in the genome sequences of CLIB 213^T^ (Galeote *et al.*[@bib17]) and ISA1307 (Mira *et al.*[@bib34]) strongly suggests that *Z. bailii* is unable to undergo mating type switching. Other genes involved in meiosis are also absent from the genome sequence of IST302, as well as in CLIB 213^T^ (Supplementary file 3), as, for example, the homologs of *S. cerevisiae* gene *IME1* that codes for a master regulator of meiosis, and of other genes involved in chromosome cohesion/recombination (*SPO13*, *SPO22*, *MER3*, *REC104*, *MLH2*, *MSH4* and *MSH5*), spore assembly (*MCP54*, *SMA1*, *SPO20*, *SPO74* and *SPO77*) or in the formation of the synaptonemal complex (*ZIP2*, *ZIP3*, *SPO16*). These genes are also absent from the genome sequence of the hybrid strain ISA1307 (Mira *et al*. [@bib34]). The life cycle of *Z. bailii* and *Z. bailii*-related strains has been investigated based on their ability to sporulate when grown in specific media (Mollapour and Piper [@bib36]; Rodrigues *et al*. [@bib50]). The peculiar formation of mitotic, but not meiotic, spores was described both in diploid *Z. bailii* strains (Mollapour and Piper [@bib36]) and in the hybrid strain ISA1307 (Rodrigues *et al*. [@bib50]). Dato *et al.* ([@bib11]) also described the presence of binucleate cells in early-stationary phase. We investigated the ability of *Z. bailii* IST302 to form spores and, surprisingly, when grown in a sporulation medium we observed the formation of protrusions between two cells (Fig. [6B--F](#fig6){ref-type="fig"}) before the development of two mitotic spores per cell (Fig. [6D and E](#fig6){ref-type="fig"}), apparently without the occurrence of karyogamy as previously described (Mollapour and Piper [@bib36]; Rodrigues *et al*. [@bib50]). Interestingly, after conjugation and formation of two spores per cell, one of the cells seems to donate each of the two spores to the other cell (Fig. [6G--I](#fig6){ref-type="fig"}) before spore release (Fig. [6J](#fig6){ref-type="fig"}). Altogether, our results suggest that *Z. bailii* IST302 haploid cells cannot undergo mating type switching, but under nutrient deprivation are able to form vegetative spores. This study provided additional information for the understanding of *Z. bailii* and *Z. bailii*-related strains life cycle complexity, although more studies involving other strains are required to disclose the peculiar life cycle of this species.

![Microscopic observation of *Z. bailii* IST302 sporulation. *Zygosaccharomyces bailii* IST302 cells were grown in a pre-sporulation medium (**A**) for 2 days and transferred to a sporulation medium (**B**--**J**). The formation of spores was observed on a Zeiss ® Axioplan microscope (×1000 magnification) after 2 (**B-D**) and 4 (**E-J**) days of incubation at 30°C.](fox025fig6){#fig6}

###### 

ORFs identified in the genomes of *Z. bailii* IST302, CLIB 213^T^ and *Z. rouxii* CBS 732^T^ encoding the homologs of *S. cerevisiae* genes involved in mating type.

  *S. cerevisiae* S288C                Biological function                                                                           *Z. bailii* IST302   *Z. bailii* CLIB 213^T^                                  *Z. rouxii* CBS 732^T^
  ------------------------------------ --------------------------------------------------------------------------------------------- -------------------- -------------------------------------------------------- ------------------------
  *MF(ALPHA)1*/2 (YPL187W / YGL089C)   Mating pheromone alpha-factor                                                                 ZBIST_1222           (Several gaps in the genome sequence at this location)   ZYRO0G08184g
  *MFA1/2* (YDR461W / YNL145W)         Mating pheromone a-factor                                                                     ZBIST_2952           \-                                                       \-
  *HMRA1*/*2* (YCR097W / *YCR096C)*    Silenced copy of a1/a2 at HMR                                                                 \-                   \-                                                       ZYRO0C18348g
  *HMLALPHA1*/*2* (YCL066W/ YCL067C)   Silenced copy of ALPHA1/2 at HML                                                              \-                   \-                                                       ZYRO0F15818g
  *MATALPHA1* (YCR040W)                Transcriptional co-activator involved in regulation of mating type specific gene expression   ZBIST_5098           BN860_00122g_l                                           ZYRO0F15840g
  *MATALPHA2* (YCR039C)                Transcriptional repressor of a-specific genes in haploids                                     \-                   \-                                                       \-
  *MATA1*                              Homeodomain protein involved in transcriptional regulation of mating type specific genes      \-                   \-                                                       \-
  *MATA2*                              Protein of unknown function; identical to the C-terminal of the MATalpha2 protein             \-                   \-                                                       \-
  *HO* (YDL227C)                       Site-specific endonuclease                                                                    ZBIST_2381           BN860_01090g_n                                           ZYRO0C10428g

Genes encoding MDR/MXR transporters {#sec3-6}
-----------------------------------

Having in mind that when *S. cerevisiae* cells are exposed to weak acids their ability to decrease the counterions' intracellular accumulation is essential to cope with their deleterious effects (reviewed by Piper *et al*. [@bib42]; Mira, Teixeira and Sá-Correia [@bib35]), we searched for transporters associated with multidrug/multixenobiotic resistance (MDR/MXR) belonging to the ATP-binding cassette (ABC) superfamily and to the Major Facilitator Superfamily (MFS) (Higgins [@bib20]; Sá-Correia *et al.*[@bib54]) proposed, or hypothesized, to be responsible for the active efflux of weak acid counterions or other toxicants. For this, homologs of several *S. cerevisiae* ABC and MFS proteins previously implicated in the MDR/MXR phenomenon were identified in the genome of *Z. bailii* IST302 (Tables [4](#tbl4){ref-type="table"}--[6](#tbl6){ref-type="table"}). Two homologs of *S. cerevisiae* ABC protein Pdr12, implicated in the active efflux of propionate, sorbate or benzoate anions (Piper *et al.*[@bib43]; Holyoak *et al.*[@bib22]), were identified in the genome of *Z. bailii* IST302 and in *Z. rouxii* CBS 732^T^ as well (Table [4](#tbl4){ref-type="table"}). Moreover, a total of four homologs of the Pdr18/Snq2 paralog pair were identified in *Z. bailii* IST302 and ISA1307 (Mira *et al.*[@bib34]), whereas only two Pdr18/Snq2 homologs were detected in *Z. bailii* CLIB 213^T^ and *Z. rouxii* (Table [4](#tbl4){ref-type="table"}). Interestingly, Pdr18 was implicated in the incorporation of ergosterol in yeast plasma membrane and found to play a role in *S. cerevisiae* tolerance to the lipophilic weak acid herbicide 2,4-D (Cabrito *et al.*[@bib8]), ethanol (Teixeira *et al.*[@bib67]) and acetic acid (Godinho, unpublished results). The paralog pair Tpo2/Tpo3 (Fernandes *et al.*[@bib14]), Aqr1 (Tenreiro *et al.*[@bib69]) and Azr1 (Tenreiro *et al.*[@bib70]) are the sole MDR/MXR transporters of the MFS so far implicated in *S. cerevisiae* tolerance to acetic acid, presumably by catalyzing the active efflux of acetate. Remarkably, *Z. bailli* IST302 genome holds only a single homolog of Tpo2 and Tpo3, and one homolog of Aqr1 and one of Azr1 (Tables [5](#tbl5){ref-type="table"} and [6](#tbl6){ref-type="table"}).

###### 

ORFs identified in the genomes of *Z. bailii* IST302, CLIB 213^T^ and *Z. rouxii* CBS 732^T^ encoding the homologs of *S. cerevisiae* MDR/MXR transporters of ABC superfamily. The homologs were organized according to the taxonomic clusters previously defined (Seret *et al.*[@bib56]).

  ABC family                    Total                                                                                             
  ----------------------------- ---------------- ---------------- ---------------- ------------- --------------- ---------------- --------
  ***S. cerevisiae* S288c**     ***PDR5***       ***SNQ2***       ***PDR12***      ***PDR11***   ***YOL075c***   ***ADP1***       **10**
                                ***PDR15***      ***PDR18***                       ***AUS1***                                     
                                ***PDR10***                                                                                       
  ***Z. bailii* IST302**        ZBIST_3521       ZBIST_0908       ZBIST_5124       \-            \-              ZBIST_3422       **12**
                                ZBIST_4874       ZBIST_0076       ZBIST_5081                                                      
                                ZBIST_4875       ZBIST_4998                                                                       
                                ZBIST_4873       ZBIST_3502                                                                       
                                ZBIST_4996                                                                                        
  ***Z. bailii* CLIB 213^T^**   BN860_00584g_q   BN860_07778g_c   BN860_00100g_t   \-            \-              BN860_01948g_i   **9**
                                BN860_04478g_f   BN860_04852g_g                                                                   
                                BN860_04500g_f                                                                                    
                                BN860_04456g_f                                                                                    
                                BN860_00276g_c                                                                                    
  ***Z. rouxii* CBS 732^T^**    ZYRO0D17710g     ZYRO0B14762g     ZYRO0F08888g     \-            ZYRO0B05588g    ZYRO0E04092g     **10**
                                ZYRO0D11858p     ZYRO0A04114g     ZYRO0F08866p                                                    
                                ZYRO0D11836p                                                                                      
                                ZYRO0D11880g                                                                                      

###### 

ORFs identified in the genomes of *Z. bailii* IST302, CLIB 213^T^ and *Z. rouxii* CBS 732^T^ encoding the homologs of *S. cerevisiae* MDR/MXR transporters of the DHA1 family from MFS. The homologs were organized according to the taxonomic clusters previously defined (Dias and Sá-Correia [@bib12]).

  DHA1 family                   Total                                                                                                                                                                          
  ----------------------------- -------------- ---------------- ------------ ---------------- ---------------- ---------------- ---------------- -------------- ------------ ---------------- ---------------- --------
  ***S. cerevisiae* S288c**     ***DTR1***     ***QDR1***       ***AQR1***   ***QDR3***       ***HOL1***       ***TPO2***       ***TPO1***       ***YHK8***     ***TPO4***   ***FLR1***       **Others**       **12**
                                               ***QDR2***                                                      ***TPO3***                                                                                      
  ***Z. bailii* IST302**        ZBIST_2327     ZBIST_5047       \-           ZBIST_0934       ZBIST_3441       ZBIST_0758       ZBIST_1684       \-             \-           ZBIST_3176       ZBIST_0204       **21**
                                               ZBIST_4184                                                                       ZBIST_1685                                   ZBIST_3177       ZBIST_4991       
                                                                                                                                                                             ZBIST_2985       ZBIST_0576       
                                                                                                                                                                             ZBIST_3175       ZBIST_3170       
                                                                                                                                                                             ZBIST_3171       ZBIST_4947       
                                                                                                                                                                             ZBIST_3173       ZBIST_5091       
                                                                                                                                                                             ZBIST_3879                        
  ***Z. bailii* CLIB 213^T^**   BN860_05512g   BN860_01244g_g   \-           BN860_08350g_c   BN860_02432g_i   BN860_04368g_h   BN860_06810g_b   \-             \-           BN860_00232g_r   BN860_07888g_d   **19**
                                                                                                                                                                             BN860_00254g_r   BN860_00232g_c   
                                                                                                                                                                             BN860_00166g_k   BN860_00188g_t   
                                                                                                                                                                             BN860_00210g_q   BN860_00122g_t   
                                                                                                                                                                             BN860_00144g_u   BN860_00276g_b   
                                                                                                                                                                             BN860_00188g_r   BN860_07470g     
                                                                                                                                                                             BN860_00232g_j                    
  ***Z. rouxii* CBS 732^T^**    ZYRO0F14652g   ZYRO0A01474g     \-           ZYRO0A03564g     ZYRO0E02288g     ZYRO0G19646g     ZYRO0B00990g     ZYRO0G16302g   \-           ZYRO0E09966g     ZYRO0E10230g     **21**
                                                                             ZYRO0F08228g                                                                                    ZYRO0E09922p     ZYRO0G15422g     
                                                                                                                                                                             ZYRO0F04642g     ZYRO0E10054g     
                                                                                                                                                                             ZYRO0E09988g     ZYRO0F02090g     
                                                                                                                                                                             ZYRO0E09900g     ZYRO0D00286g     
                                                                                                                                                                             ZYRO0B16808p     ZYRO0F02178p     
                                                                                                                                                                             ZYRO0A13618g                      

###### 

ORFs identified in the genomes of *Z. bailii* IST302, CLIB 213^T^ and *Z. rouxii* CBS 732^T^ encoding the homologs of *S. cerevisiae* MDR/MXR transporters of the DAG family from MFS. The homologs were organized according to the taxonomic clusters previously defined (Dias and Sá-Correia [@bib12]).

  DAG family                                                                                                                                                                             
  ----------------------------- ---------------- ---------------- ---------------- ------------- -------------- ---------------- ------------ ------------ ---------------- ------------ --------
  **Subfamily**                 **DHA2**         **ARN**          **GEX**          **Total**                                                                                             
  ***S. cerevisiae* S288c**     ***SGE1***       ***VBA1***       ***VBA4***       ***ATR1***    **YOR378W**    **Others**       ***ARN4***   ***ARN3***   ***ARN1***       ***GEX1***   **16**
                                ***AZR1***       ***VBA2***                        **YMR279C**                                                             ***ARN2***       ***GEX2***   
                                ***VBA3***                                                                                                                                               
                                ***VBA5***                                                                                                                                               
  ***Z. bailii* IST302**        ZBIST_3880       ZBIST_0896       ZBIST_0616       \-            ZBIST_4630     ZBIST_3876       \-           ZBIST_4766   ZBIST_2552       \-           **12**
                                ZBIST_0332       ZBIST_3511                                                                                                ZBIST_4135                    
                                                 ZBIST_0774                                                                                                                              
                                                 ZBIST_0773                                                                                                                              
  ***Z. bailii* CLIB 213^T^**   BN860_00188g_k   BN860_07514g_c   BN860_01134g_l   \-            BN860_01552g   BN860_00386g_g   \-           \-           BN860_04984g_f   \-           **11**
                                BN860_10814g_c   BN860_00320g_q                                                                                            BN860_00144g_p                
                                                 BN860_04742g_g                                                                                                                          
                                                 BN860_04720g_f                                                                                                                          
  ***Z. rouxii* CBS 732^T^**    \-               ZYRO0G03234g     ZYRO0C01430g     \-            \-             \-               \-           \-           ZYRO0G07414g     \-           **6**
                                                 ZYRO0A00330g                                                                                                                            
                                                 ZYRO0G21868g                                                                                                                            
                                                 ZYRO0G21890p                                                                                                                            

The *Z. bailii* strains tested are remarkably more tolerant to the lipophilic weak acid herbicides 2,4-D and MCPA, as well as to the herbicide alachlor of a different chemical family, when compared to *S. cerevisiae*. The susceptibility of *S. cerevisiae* to 2,4-D and MCPA was found to significantly decrease in a mutant with the *PDR5* gene deleted (Teixeira and Sá-Correia [@bib68]). This gene encodes a plasma membrane ATP transporter involved in pleiotropic drug resistance. Interestingly, five homologs of the Pdr5/Pdr15/Pdr10 taxonomic cluster were identified in *Z. bailii* IST302 (Table [4](#tbl4){ref-type="table"}) three of them are likely to be the result of two gene duplications (ZBIST_4873, ZBIST_4874 and ZBIST_4875). Moreover, in *Z. bailii* IST302 two paralogs are predicted to encode Tpo1-like transporters (Table [5](#tbl5){ref-type="table"}). The MFS transporter Tpo1 was also associated with *S. cerevisiae* tolerance to 2,4-D and MCPA (Teixeira and Sá-Correia [@bib68]). As highlighted before (Mira *et al.*[@bib34]), the total number of MFS-MDR/MXR homologs identified in *Z. bailii* and *Z. rouxii* is remarkably higher when compared with *S. cerevisiae* S288c genome. However, one of the most remarkable differences in the number of genes encoding these transporters between these *Zygosaccharomyces* species and *S. cerevisiae* is the number of Flr1 homologs. *Zygosaccharomyces bailii* and *Z. rouxii* do possess seven Flr1-like transporters and the hybrid strain ISA1307 holds 12 homologs of *S. cerevisiae* Flr1 (Mira *et al.*[@bib34]). *Saccharomyces cerevisiae* Flr1 is responsible for resistance to several drugs, such as the fungicide fluconazole (Alarco *et al.*[@bib1]; Brôco *et al.*[@bib6]) and mancozeb (Teixeira *et al.*[@bib66]). However, no significant difference in susceptibility to agricultural fungicide mancozeb could be registered in *Z. bailii* and *S. cerevisiae* strains. Interestingly, the hybrid strain ISA1307 is significantly more tolerant to all the antifungal drugs tested than *Z. bailii* IST302 or *S. cerevisiae* BY4741. Among the antifungal drugs tested, *Z. bailii* IST302 is only mildly more tolerant to fluconazole and miconazole when compared to *S. cerevisiae*. The antifungal activity of azole drugs relies on the inhibition of ergosterol biosynthetic enzymes (Hitchcock *et al.*[@bib21]; White, Marr and Bowden [@bib76]). Differences in the basal content of ergosterol of *Z. bailii* and *S cerevisiae* strains (Lindberg *et al.*[@bib29]), among other reasons (White, Marr and Bowden [@bib76]; Prasad, Shah and Rawal [@bib45]), could justify the moderate differences observed in the susceptibility phenotypes of these two yeast species.

Although interesting, this genomic study involving the comparison of the number of MDR/MXR transporters homologous to those functionally related with resistance to weak acids and other inhibitory compounds in *S. cerevisiae* has limitations. In fact, the specificity and efficiency of these transporters as well as their expression levels have to be first investigated in *Z. bailii* cells challenged with those compounds before taking conclusions. Moreover, it was reported that other mechanisms are likely involved in *Z. bailii* tolerance to drugs/xenobiotics. For example, studies suggest that *Z. bailii* tolerance to acetic acid is related with a number of alternative physiological strategies that include the capacity to tolerate short-term intracellular pH changes (Arneborg, Jespersen and Jakobsen [@bib2]; Dang *et al.*[@bib9]), to co-consume acetic acid and glucose (Sousa *et al.*[@bib60]; Guerreiro, Mira and Sá-Correia [@bib18]; Rodrigues *et al.*[@bib51]) and to exhibit a reduced permeability of plasma membrane to acetic acid due to the higher basal level of complex sphingolipids (Lindahl *et al.*[@bib28]).

*Zygosaccharomyces bailii* IST302 does not form cell aggregates {#sec3-7}
---------------------------------------------------------------

Two of the most striking differences between *Z. bailii* IST302 and CLIB 213^T^ are the morphology of their colonies (Fig. [7A](#fig7){ref-type="fig"}) and the formation of large cellular aggregates (Fig. [7B](#fig7){ref-type="fig"}) by *Z. bailii* CLIB 213^T^. When cultivated either in minimal or rich media, CLIB 213^T^ shows a rough colony morphology (Fig. [7A](#fig7){ref-type="fig"}) and forms cell aggregates that are visible with naked eye and under bright-field microscopy (Fig. [7B](#fig7){ref-type="fig"}). Differently, *Z. bailii* IST302 shows regular and smooth colonies (Fig. [7A](#fig7){ref-type="fig"}) and absence of cellular aggregation (Fig. [7B](#fig7){ref-type="fig"}), which makes this strain very easy to manipulate for physiological studies when compared to the reference CLIB 213^T^ strain*.* In order to investigate whether this type of cellular aggregation was due to flocculation, the dispersion of *Z. bailii* CLIB 213^T^ cell aggregates was attempted by suspending cells in 0.05 M EDTA (pH 8.0) (Stratford [@bib62]), but no significant dispersion of the aggregates was observed in the presence of the chelating agent, nor the addition of CaCl~2~ significantly increased cellular aggregation (Fig. [7B](#fig7){ref-type="fig"}). This result suggests that the aggregation of *Z. bailii* CLIB 213^T^ cells results from a Ca^2+^-independent mechanism. *Zygosaccharomyces bailii* IST302 and the *Z. bailii* hybrid strain ISA1307 were found to be non-flocculent yeast strains given that the suspension of each of these strains in 0.01 M CaCl~2~ did not induce a flocculation phenotype (Stratford [@bib62]). *Zygosaccharomyces bailii* CLIB 213^T^ was also cultivated in YP medium containing fructose or mannose instead of glucose (Soares *et al*. [@bib57]), but cellular aggregation was still detected (results not shown). Interestingly, the cellular aggregation phenotype of CLIB 213^T^ is similar to that described and identified by Ratcliff *et al*. ([@bib48]) as the 'snowflake' yeast phenotype which in *S. cerevisiae* was found to be related to specific mutations in Ace2 transcription factor (Ratcliff *et al.*[@bib49]), required for septum destruction after cytokinesis. The comparative analysis of *Z. bailii* genes homologous to *S. cerevisiae* genes involved in daughter cell separation as well as other genes involved in cell budding or morphogenesis and whose deletion led to abnormal budding pattern or the formation of cell aggregates was performed in both IST302 and CLIB 213^T^ strains (Table [7](#tbl7){ref-type="table"}). No gene duplications or eliminations were detected, but a closer look into the protein sequences of the homolog of *S. cerevisiae* Ace2 transcription factor, ZBIST_2164 and BN860_01596g_h from *Z. bailii* IST302 and CLIB 213^T^, respectively, allowed the identification of five amino acid substitutions, of which three involving serine/threonine residues. The pairwise alignment of *Z. bailii* IST302 and CLIB 213^T^ Ace2 homologs is available in Supplementary file 4 (Supporting Information). It was recently reported that non-synonymous mutations located or adjacent to Ace2 zinc finger-binding domain in the C-terminal region of the protein are responsible for the *S. cerevisiae* 'snowflake' phenotype (Ratcliff *et al.*[@bib49]). Although the comparison of Ace2 homologs from IST302 and CLIB 213^T^ did not identify amino acid substitutions in the C-terminal, one cannot discard the hypothesis that the identified mutations might also be involved in the aggregation phenotype shown by CLIB 213^T^. Nevertheless, other proteins can also have a role in the aggregation phenotype. For example, *Z. bailii* IST302 and CLIB 213^T^ homologs of Cbk1 and Cts1, which in *S. cerevisiae* encode, respectively, a serine/threonine kinase involved in the regulation of Ace2 localization and activity and an endochitinase required for cell separation after mitosis whose transcriptional activation is mediated by Ace2, also show differences in their amino acid sequences (Table [7](#tbl7){ref-type="table"}, Supplementary file 4).

![Microscopic observation of the morphology of the colonies (**A**) and cells (**B**) of *Z. bailii* IST302, *Z. bailii* CLIB 213^T^ and ISA1307 hybrid strain. (**A**) Colonies of *Z. bailii* IST302, *Z. bailii* CLIB 213^T^ and ISA1307 hybrid strain cells plated onto YPD plates and grown for 5 days were observed using a stereomicroscope and photographed. (**B**) Cells of *Z. bailii* IST302, *Z. bailii* CLIB 213^T^ and the hybrid strain ISA1307 grown in YPD were observed on a Zeiss® Axioplan microscope (×400 magnification) and photographed.](fox025fig7){#fig7}

###### 

*Zygosaccharomyces bailii* homologs of *S. cerevisiae* genes involved in cell aggregation.

  *S. cerevisiae* gene   Phenotype of *S. cerevisiae* null mutant (reference)                                                                                                                                 *Z. bailii* IST302   *Z. bailii* CLIB 213^T^   IST302 and CLIB 213^T^ protein sequence alignment identity (%)
  ---------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ -------------------- ------------------------- ----------------------------------------------------------------
  *ACE2*/*SWI5*          Abnormal budding pattern (Voth *et al.*[@bib73])                                                                                                                                     ZBIST_2164           BN860_01596g_h            99.3
  *CBK1*                 Abnormal budding pattern (Voth *et al.*[@bib73])                                                                                                                                     ZBIST_0511           BN860_14928g1_1           97.3
                         Cells form aggregates and display cell separation defects (Nelson *et al.*[@bib38])                                                                                                                                                 
                         Cells form large aggregates that do not easily dissociate with a micro-manipulator or EDTA treatment but separate upon the expression of chitinase (CTS1) (Racki *et al.*[@bib47])                                                  
  *BUD4*                 Abnormal budding pattern (Voth *et al.*[@bib73])                                                                                                                                     ZBIST_1783           BN860_04500g_e            99.2
  *MOB2*                 Cells form aggregates and display cell separation defects (Nelson *et al.*[@bib38])                                                                                                  ZBIST_4670           BN860_00694g_d            99.6
  *TAO3*                 Cells form aggregates and display cell separation defects (Nelson *et al.*[@bib38])                                                                                                  ZBIST_4175           BN860_01046g_h1           99.5
  *KIC1*                 Cells form aggregates and display cell separation defects (Nelson *et al.*[@bib38])                                                                                                  ZBIST_1895           BN860_02850g1_1           99.5
  *SOG2*                 Cells form aggregates and display cell separation defects (Nelson *et al.*[@bib38])                                                                                                  ZBIST_1556           BN860_05226g_b            99.8
  *HYM1*                 Cells form aggregates and display cell separation defects (Nelson *et al.*[@bib38])                                                                                                  ZBIST_3061           BN860_02454g_d            100
                         Clumps of cells, possibly due to failed septum removal (Bidlingmaier *et al.*[@bib3])                                                                                                                                               
  *CTS1*                 Cell separation blocked (Kuranda and Robbins [@bib26])                                                                                                                               ZBIST_3250           BN860_01904g_j            98.2

In conclusion, this study provides relevant information on the genome sequence and annotation of *Z. bailii* IST302 that, contrasting with the reference strain *Z. bailii* CLIB 213^T^, does not form cell aggregates, thus being amenable to physiological studies. This haploid strain is easy to transform (Palma *et al.*[@bib40]) and therefore eventually suitable to be engineered exploring genome editing tools. For these reasons, *Z. bailii* IST302 is proposed as an attractive strain to be used for enlightening the mechanisms underlying its remarkable tolerance to stress induced by acetic acid and other weak acid food preservatives or for the exploitation of its potential use as microbial cell factory. The availability of *Z. bailii* IST302 genome sequence and annotation is an essential step towards the achievement of these important objectives.

Supplementary Material
======================

###### 

Supplementary data are available at *[FEMSYR](https://academic.oup.com/femsyr)* online.

###### 

Click here for additional data file.

We thank Dr Cláudia L. da Silva, Ricardo Pereira and António Soure from iBB for helpful suggestions regarding flow cytometry data acquisition and interpretation.

SUPPLEMENTARY DATA {#sec4}
==================

Supplementary data are available at *[FEMSYR](https://academic.oup.com/femsyr)* online.

FUNDING {#sec5}
=======

This work was partially supported by Institute for Bioengineering and Biosciences - iBB \[BBI14\]. Funding received by iBB from Fundação para a Ciência e a Tecnologia (FCT) \[UID/BIO/04565/2013\] and from Programa Operacional Regional de Lisboa 2020 \[Project N. 007317\] is also acknowledged. MP is the recipient of a post-doctoral fellowship \[SFRH/BPD/73306/2010\] awarded by FCT.

***Conflict of interest.*** None declared.
