Brettanomyces bruxellensis SSU1Haplotypes Confer Different Levels of Sulfite Tolerance When Expressed in aSaccharomyces cerevisiae SSU1Null Mutant

Abstract

The addition of SO 2 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 SO 2 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 SO 2 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 SO 2 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 SO 2 tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO 2 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.IMPORTANCE Brettanomyces 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 SO 2 , with relatively high concentrations of this antimicrobial needed for their control. Although SO 2 tolerance has been studied in several organisms and particularly in S. cerevisiae, little is known about the mechanisms that confer SO 2 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 SO 2 tolerance. Our results suggest that a combination of BbSSU1 haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO 2 tolerances observed for different B. bruxellensis strains.

New genome assemblies reveal patterns of domestication and adaptation across Brettanomyces (Dekkera) species

“…bruxellensis and to a lesser extent B. anomalus, are the main species encountered during wine and beer fermentation and has led to the majority of Brettanomyces research focusing only on these two species. The initial assembly of the triploid B. bruxellensis strain AWRI1499 [18] has enabled genomics to facilitate research on this organism [19][20][21][22][23]. Subsequent efforts have seen the B. bruxellensis genome resolved to chromosome-level scaffolds [24].…”

Genome sequencing, annotation and exploration of the SO2-tolerant non-conventional yeast Saccharomycodes ludwigii

“…Tolerance to SO 2 in S. cerevisiae has been largely attributed to the activity of the sulfite export pump Ssu1 [ 21 , 22 , 69 ], which was also found to influence tolerance to this preservative in the more tolerant strain Brettanomyces bruxellensis [ 23 ]. Notably, the genome of S. ludwigii UTAD17 was found to encode four genes with a strong similarity (above 45% at the amino acid level) with the efflux pump ScSsu1: SCLUD1.g608, SCLUD1.g608b, SCLUD1.g612, SCLUD1.g612b) (supplementary figure S 5 ).…”

“…To counter-act the deleterious effect of intracellular accumulation of SO 2 , S. cerevisiae relies on the activity of the sulfite plasma membrane transporter Ssu1, which is believed to promote the extrusion of metabisulfite [ 21 , 22 ]. The high tolerance to SO 2 of Brettanomyces bruxellensis , another relevant wine spoilage species, was also associated to the activity of Ssu1 [ 23 ], however, in S. ludwigii no such similar transporter has been described until thus far. In fact, the molecular traits underlying the high tolerance to SO 2 of S. ludwigii remain largely unchacterized.…”

Biodiversity among Brettanomyces bruxellensis Strains Isolated from Different Wine Regions of Chile: Key Factors Revealed about Its Tolerance to Sulphite

“…Five out of 15 strains tested were susceptible to the lowest SO 2 concentration (0.3 mg L −1 ). One of the most widely known mechanisms of SO 2 tolerance involves the activity of the SSU1 sulphite pump [32,33], a member of the Tellurite-resistance/dicarboxylate transporter (TDT) family. Diversity in sulphite tolerance has been found in strains from different geographical areas and with varying physiological patterns of behaviour [34,35], which suggests that this characteristic can be determined by quantitative factors.…”

“…However, this cannot apply to processes contaminated with L-2676 or L-2679 clonal groups. The existence of allotriploidy and genotype diversity in B. bruxellensis has been linked to high tolerance to SO 2 [31][32][33][34][35][36][37][38], and has been attributed to the selection that occurs when adding this antimicrobial to the wide range of fermented beverages. If allotriploidy is the key factor in these two strains, this is a matter that requires further investigation.…”