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

The addition of SO 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 exhibit significant SO tolerance, largely driven by the efflux pump Ssu1p. One of the key yeast species responsible for wine spoilage is , which also exhibits strain-dependent SO tolerance, although this occurs via unknown mechanisms. To evaluate the factors responsible for the differential sulfite tolerance observed in strains, we employed a multifaceted approach to examine both expression and allelic differences in the gene. Transcriptomic analysis following exposure to SO highlighted different inducible responses in two strains. It also revealed disproportionate transcription of one putative haplotype in both genetic backgrounds. Here, we confirm the functionality of by complementation of a null mutant in a wine strain. The expression of four distinct haplotypes in the Δ mutant revealed up to a 3-fold difference in conferred SO tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO 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 allele that encodes a more efficient Ssu1p transporter may represent one mechanism that contributes to differences in sulfite tolerances between strains. 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 SO , with relatively high concentrations of this antimicrobial needed for their control. Although SO tolerance has been studied in several organisms and particularly in , little is known about the mechanisms that confer SO tolerance in Here, we confirmed the functionality of the sulfite efflux pump encoded by and determined the efficiencies of four different haplotypes. Gene expression analysis showed greater expression of the haplotype conferring greater SO tolerance. Our results suggest that a combination of haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO toleranc