Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic manipulations and physiological studies

Abstract

Zygosaccharomyces bailii is one of the most problematic spoilage yeast species found in the food and beverage industry particularly in acidic products, due to its exceptional resistance to weak acid stress. This article describes the annotation of the genome sequence of Z. bailii IST302, a strain recently proven to be amenable to genetic manipulations and physiological studies. The work was based on the annotated genomes of strain ISA1307, an interspecies hybrid between Z. bailii and a closely related species, and the Z. bailii reference strain CLIB 213T. The resulting genome sequence of Z. bailii IST302 is distributed through 105 scaffolds, comprising a total of 5142 genes and a size of 10.8 Mb. Contrasting with CLIB 213T, strain IST302 does not form cell aggregates, allowing its manipulation in the laboratory for genetic and physiological studies. Comparative cell cycle analysis with the haploid and diploid Saccharomyces cerevisiae strains BY4741 and BY4743, respectively, suggests that Z. bailii IST302 is haploid. This is an additional trait that makes this strain attractive for the functional analysis of non-essential genes envisaging the elucidation of mechanisms underlying its high tolerance to weak acid food preservatives, or the investigation and exploitation of the potential of this resilient yeast species as cell factory.

Keywords: Zygosaccharomyces bailii; cellular aggregation; food spoilage yeasts; genome sequence; weak acid tolerance.

Figure 1.

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. (2013).

Figure 2.

Zygosaccharomyces 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 × 10⁵ 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.

Figure 3.

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).

Figure 4.

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.

Figure 5.

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 2010).

Figure 6.

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.

Figure 7.

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.