Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species

Abstract

Background: Aureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans.

Results: The 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus.

Conclusions: The differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae. The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.

Figure 1

The Aureobasidium pullulans varieties. A. Phylogram showing the phylogenetic relationships of the four A. pullulans varieties and their phylogenetic position, inferred from super alignment of selected fungal proteomes. Chi²-based branch supports are shown, calculated according to the approximate Likelihood-Ratio Test, as implemented in Phyml 3.0. B. Representative images of one-month-old cultures of the four A. pullulans varieties (as indicated) on malt extract agar and microscopy images of cultures after one week of growth on malt extract agar blocks.

Figure 2

The shared, unique and duplicated proteins of the Aureobasidium pullulans varieties. A. Unique and shared protein families, as determined by the Markov clustering algorithm. B. Proteins shared between individual varieties, as determined by all-against-all blastp. C. Proteins present in at least two copies in the proteome of each of the four A. pullulans varieties. P, A. pullulans var. pullulans; S, A. pullulans var. subglaciale; N, A. pullulans var. namibiae; M, A. pullulans var. melanogenum.

Figure 3

Aquaporin genes in Aureobasidium pullulans and other fungi. A. Protein tree of aquaporin-like genes from the four A. pullulans varieties and other fungi. The tree with GenBank accession numbers is available as Additional file 6. Colours correspond to previously recognised phylogenetic groups [116], and aquaporin-like proteins of the A. pullulans varieties are marked in red. B. Histogram showing the number of aquaporins in the four A. pullulans varieties, as compared to the other fungal species reported by Xu et al. [116].

Figure 4

Protein trees of the various membrane transporters of Na ⁺ and K ⁺ . The protein trees are labelled according to the names of homologues from S. cerevisiae (except Acu transporters, which have no S. cerevisiae homologues). The trees (except for Acu) were rooted with homologous proteins from C. neoformans (Trk: [GenBank:XP_570017], [GenBank:XP_569339]; Tok: [GenBank:XP_568987], [GenBank:XP_568988]; Nha: [GenBank:XP_569560]; Ena: [GenBank:XP_572412], [GenBank:XP_568029], [GenBank:XP_570160]; Pho: [GenBank:XP_568082]) and root locations marked with an arrow. In addition to genes from A. pullulans, homologues from related fungi were used, as labelled with the fungal species name and GenBank accession number (H. werneckii, M. graminicola, S. cerevisiae), or the Joint Genome Institute Genome Portal protein ID (all of the rest). Putative gene duplications leading to the present diversity of these genes in A. pullulans are indicated by double arrows. ApP, A. pullulans var. pullulans; ApS, A. pullulans var. subglaciale; ApN, A. pullulans var. namibiae; ApM, A. pullulans var. melanogenum.

Figure 5

Kr distances between the genomes of Aureobasidium pullulans and selected pairs of genomes of Saccharomyces cerevisiae, Saccharomyces mikatae , and Saccharomyces kudriavzevii . Distances are represented by the lengths of the horizontal bars.

Figure 6

Configuration of the homothallic Aureobasidium species MAT loci and adjacent genes. Names of the four new Aureobasidium species are indicated above the gene models. Black boxes, exons; arrows, direction of transcription of the gene.