Brettanomyces bruxellensis SSU1 Haplotypes Confer Different Levels of Sulfite Tolerance When Expressed in a Saccharomyces cerevisiae SSU1 Null Mutant

Abstract

The addition of SO₂ is practiced in the wine industry to mitigate the risk of microbial spoilage and to extend wine shelf-life. Generally, this strategy does not interfere with primary alcoholic fermentation, as wine strains of Saccharomyces cerevisiae exhibit significant SO₂ tolerance, largely driven by the efflux pump Ssu1p. One of the key yeast species responsible for wine spoilage is Brettanomyces bruxellensis, which also exhibits strain-dependent SO₂ tolerance, although this occurs via unknown mechanisms. To evaluate the factors responsible for the differential sulfite tolerance observed in B. bruxellensis strains, we employed a multifaceted approach to examine both expression and allelic differences in the BbSSU1 gene. Transcriptomic analysis following exposure to SO₂ highlighted different inducible responses in two B. bruxellensis strains. It also revealed disproportionate transcription of one putative BbSSU1 haplotype in both genetic backgrounds. Here, we confirm the functionality of BbSSU1 by complementation of a null mutant in a S. cerevisiae wine strain. The expression of four distinct BbSSU1 haplotypes in the S. cerevisiae ΔSSU1 mutant revealed up to a 3-fold difference in conferred SO₂ tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO₂ tolerance. Protein modeling of two haplotypes which differed in two amino acid residues suggested that these substitutions affect the binding of Ssu1p ligands near the channel opening. Taken together, preferential transcription of a BbSSU1 allele that encodes a more efficient Ssu1p transporter may represent one mechanism that contributes to differences in sulfite tolerances between B. bruxellensis strains.IMPORTANCEBrettanomyces bruxellensis is one of the most important wine spoilage microorganisms, with the use of sulfite being the major method to control spoilage. However, this species displays a wide intraspecies distribution in sulfite tolerance, with some strains capable of tolerating high concentrations of SO₂, with relatively high concentrations of this antimicrobial needed for their control. Although SO₂ tolerance has been studied in several organisms and particularly in S. cerevisiae, little is known about the mechanisms that confer SO₂ tolerance in B. bruxellensis Here, we confirmed the functionality of the sulfite efflux pump encoded by BbSSU1 and determined the efficiencies of four different BbSSU1 haplotypes. Gene expression analysis showed greater expression of the haplotype conferring greater SO₂ tolerance. Our results suggest that a combination of BbSSU1 haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO₂ tolerances observed for different B. bruxellensis strains.

Keywords: SO2; Ssu1p; allele specific expression; transcriptome; wine; yeast.

FIG 1

Population and transcriptional responses of B. bruxellensis to sulfite treatment. (A) Relative change in culturability (CFU) for triplicate cultures of strains AWRI1499 and AWRI1613 treated with SO₂; boxes represent range with median. (B) Venn diagram summarizing open reading frames (ORFs) and novel transcriptionally active regions (nTARs) significantly upregulated (UP) or downregulated (DOWN) 2 h after SO₂ treatment for both strains.

FIG 2

Skewed transcript abundance of BbSSU1 allele. Major allele frequency calculated at each polymorphic SNP across BbSSU1 (g80.t1) open reading frame for genomic (blue) and transcriptomic (red) data sets. Box plots represent 12 polymorphic sites for triploid AWRI1499, 11 for diploid AWRI1613, and 11 for UniFG14 (ploidy unknown) across all biological replicates (n = 3 for RNA-seq, n = 1 for genomic data).

FIG 3

(A) Phylogenetic tree for B. bruxellensis SSU1 haplotypes. AWRI1499 (red) has two haplotypes, one being present in two copies; AWRI1613 (blue) has two haplotypes; and AWRI2804 (green) has only one haplotype. (B) Amino acid sequences of Ssu1 proteins encoded by the four B. bruxellensis SSU1 haplotypes. Differences in amino acid residues are shown using AWRI1499_SSU1_hap1 as the template. Cytoplasmic domains (yellow bar), transmembrane domains (blue bar), and noncytoplasmic domains (orange bar) were predicted using SPOCTOPUS.

FIG 4

(A) Multicopy expression of B. bruxellensis SSU1 haplotypes in S. cerevisiae. SSU1 haplotypes were inserted in the multicopy vector pCV3 and expressed in S. cerevisiae strain AWRI1631 ΔSSU1. The results shown represent the average of the results from three replicates per strain repeated twice as independent experiments. (B) Single-copy expression of B. bruxellensis SSU1 haplotypes in S. cerevisiae. SSU1 haplotypes were expressed in S. cerevisiae strain AWRI1631 ΔSSU1 under the control of the native S. cerevisiae promoter. Fisher’s LSD test was used to determine significant differences (P < 0.05).

FIG 5

(A) Evaluation of Ssu1p amino acid substitutions between AWRI1613_SSU1_hap2 and AWRI1499_SSU1_hap2. (B) Evaluation of Ssu1p amino acid substitutions between AWRI1499_SSU1_hap1 and AWRI1499_SSU1_hap2. Fisher’s LSD test was used to determine significant differences (P < 0.05).

FIG 6

Superposition of average protein structures for AWRI1499_SSU1_hap2 (pink) and AWRI1613_SSU1_hap2 (blue). Amino acid residues differing between both haplotypes and relevant protein domains are shown.

FIG 7

HSO₃⁻ docking simulations for protein structures of AWRI1499_SSU1_hap2 (A) and AWRI1613_SSU1_hap2 (B). Protein structures are shown from the cytoplasm with protein molecular surface colored by Coulombic electrostatic potential (blue, positive charge; red, negative charge). HSO₃⁻ is colored by atom (red, oxygen; white, hydrogen; yellow, sulfur).