Retrotransposon-Mediated Duplication of SSU1 in High SO2-Tolerant Brettanomyces bruxellensis Winery Isolates

Abstract

Background and Aims

Brettanomyces bruxellensis is an industrially relevant yeast and a major spoilage organism in wine, where tolerance to sulphur dioxide (SO₂), the primary preservative used for its control, varies among strains. Although laboratory evolution experiments have shown that polyploid B. bruxellensis can rapidly acquire increased SO₂ tolerance through genomic variation and SSU1 duplication, it remains unclear whether similar genetic adaptations drive SO₂ tolerance in winery populations. To address this question, B. bruxellensis strains were isolated from wineries across Australia and subjected to phenotypic characterisation of SO₂ tolerance and detailed genomic analyses to investigate the genetic basis of SO₂ tolerance in industry isolates.

Methods and Results

Twenty-six isolates from Australian wineries were phenotypically characterised and sequenced using long-read technology. Isolates showed a wide range of SO₂ tolerance that correlated with phylogenetic clade and ploidy. Haplotype phasing of SSU1, a sulphite efflux pump linked to SO₂ tolerance, identified nine distinct haplotypes, including the previously described high-tolerance H1 allele. Highly tolerant strains carried duplications of H1, frequently associated with retrotransposon insertions and chromosomal rearrangements at the SSU1 locus. Comparative analyses with laboratory-evolved strains confirmed that retrotransposons facilitated the acquisition of additional SSU1 copies.

Conclusions

Structural variation at the SSU1 locus, particularly duplication of the high-tolerance H1 haplotype, is associated with increased SO₂ tolerance in winery isolates of B. bruxellensis. The similarities between industry strains and laboratory-evolved strains suggest that comparable adaptive mechanisms operate in winery environments. These results improve our understanding of how this spoilage yeast evolves sulphite tolerance and persists in wineries.