Genomic characterization of polyextremotolerant black yeasts isolated from food and food production environments

Abstract

Black yeasts have been isolated from acidic, low water activity, and thermally processed foods as well as from surfaces in food manufacturing plants. The genomic basis for their relative tolerance to food-relevant environmental stresses has not been well defined. In this study, we performed whole genome sequencing (WGS) on seven black yeast strains including Aureobasidium (n=5) and Exophiala (n=2) which were isolated from food or food production environments. These strains were previously characterized for their tolerance to heat, hyperosmotic pressure, high pressure processing, hypochlorite sanitizers, and ultraviolet light. Based on the WGS data, three of the strains previously identified as A. pullulans were reassigned as A. melanogenum. Both haploid and diploid A. melanogenum strains were identified in this collection. Single-locus phylogenies based on beta tubulin, RNA polymerase II, or translation elongation factor protein sequences were compared to the phylogeny produced through SNP analysis, revealing that duplication of the fungal genome in diploid strains complicates the use of single-locus phylogenetics. There was not a strong association between phylogeny and either environmental source or stress tolerance phenotype, nor were trends in the copy numbers of stress-related genes associated with extremotolerance within this collection. While there were obvious differences between the genera, the heterogenous distribution of stress tolerance phenotypes and genotypes suggests that food-relevant black yeasts may be ubiquitous rather than specialists associated with particular ecological niches. However, further evaluation of additional strains and the potential impact of gene sequence modification is necessary to confirm these findings.

Keywords: Aureobasidium; Exophiala; food mycology; genome duplication; single-gene phylogeny.

Figure 1

Phylogenetic relationships among Aureobasidium strains inferred from SNP alignment. Bootstrap values observed among 1,000 replicates are indicated. The phylogenetic tree was midpoint rooted and the scale bar denotes the number of nucleotide substitutions per site.

Figure 2

Phylogenetic relationships of Exophiala strains inferred from SNP alignment. Bootstrap values observed among 1,000 replicates are indicated. The phylogenetic tree was midpoint rooted and the scale bar denotes the number of nucleotide substitutions per site.

Figure 3

Neighbor-joining phylogenies based on protein sequences for (A) beta tubulin, (B) RNA polymerase II RPB2, and (C) translation elongation factor TEF1 in eight black yeast strains and five other black yeast type strains (AP, A. pullulans; AM, A. melanogenum; ED, E. dermatitidis; EP, E. phaeomuriformis; SC, Saccharomyces cerevisiae).

Figure 4

SNP-based phylogeny of black yeast isolated from food or food production environments (left) and their relative stress tolerance to diverse food processing conditions (right).

Figure 5

Distribution of unique protein coding genes or pseudogenes in Aureobasidium genomes.

Figure 6

Number of gene homologues in food-relevant black yeast (per haploid genome). (A) Oxidative stress response genes: catalase (left) and copper, zinc superoxide dismutase (right); (B) Alkali-metal cation transporters: potassium transport protein (left) and cation efflux system proteins (right); (C) Aquaporins: aquaglyceroporin (left) and aquaporin (right); (D) Melanin biosynthesis genes: laccase (left) and polyketide synthase (right); (E) Genes in biosynthesis of compatible solutes: α, α-trehalose-phosphate synthase (left) and NAD-dependent glycerol-3-phosphate dehydrogenase (right); (F) Components of the high-osmolarity glycerol pathway: high osmolarity signaling protein (left) and mitogen-activated protein kinase (right).

Figure 7

Correlation between the number of stress-related genes in food-relevant black yeast (left) and between gene number and stress tolerance phenotype (right). *p < 0.05; **p < 0.01; ***p < 0.001.