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Genome and Transcriptome Sequencing of the Halophilic Fungus Wallemia Ichthyophaga: Haloadaptations Present and Absent

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Genome and Transcriptome Sequencing of the Halophilic Fungus Wallemia Ichthyophaga: Haloadaptations Present and Absent

Janja Zajc et al. BMC Genomics.

Abstract

Background: The basidomycete Wallemia ichthyophaga from the phylogenetically distinct class Wallemiomycetes is the most halophilic fungus known to date. It requires at least 10% NaCl and thrives in saturated salt solution. To investigate the genomic basis of this exceptional phenotype, we obtained a de-novo genome sequence of the species type-strain and analysed its transcriptomic response to conditions close to the limits of its lower and upper salinity range.

Results: The unusually compact genome is 9.6 Mb large and contains 1.67% repetitive sequences. Only 4884 predicted protein coding genes cover almost three quarters of the sequence. Of 639 differentially expressed genes, two thirds are more expressed at lower salinity. Phylogenomic analysis based on the largest dataset used to date (whole proteomes) positions Wallemiomycetes as a 250-million-year-old sister group of Agaricomycotina. Contrary to the closely related species Wallemia sebi, W. ichthyophaga appears to have lost the ability for sexual reproduction. Several protein families are significantly expanded or contracted in the genome. Among these, there are the P-type ATPase cation transporters, but not the sodium/ hydrogen exchanger family. Transcription of all but three cation transporters is not salt dependent. The analysis also reveals a significant enrichment in hydrophobins, which are cell-wall proteins with multiple cellular functions. Half of these are differentially expressed, and most contain an unusually large number of acidic amino acids. This discovery is of particular interest due to the numerous applications of hydrophobines from other fungi in industry, pharmaceutics and medicine.

Conclusions: W. ichthyophaga is an extremophilic specialist that shows only low levels of adaptability and genetic recombination. This is reflected in the characteristics of its genome and its transcriptomic response to salt. No unusual traits were observed in common salt-tolerance mechanisms, such as transport of inorganic ions or synthesis of compatible solutes. Instead, various data indicate a role of the cell wall of W. ichthyophaga in its response to salt. Availability of the genomic sequence is expected to facilitate further research into this unique species, and shed more light on adaptations that allow it to thrive in conditions lethal to most other eukaryotes.

Figures

Figure 1
Figure 1
Wallemia ichthyophaga . A. Phylogram showing the phylogenetic origin of W. ichthyophaga, inferred from a 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. Colonies of W. ichthyophaga on yeast nitrogen base medium with 25% NaCl (w/v), and a microscopic image showing the characteristic meristematic clumps (arrow) formed by isodiametric growth of groups of thick-walled cells next to the cubic crystals of halite (NaCl).
Figure 2
Figure 2
Circular representation of the Wallemia ichthyophaga genome. The following data are shown (from outside, in): (a) Differential expression as log2 ratio of expression at high salinity (30% NaCl [w/v]) versus low salinity (10% NaCl [w/v]), with increased expression in red and decreased in green (scale −2 to 2); genes with false discovery rates larger than 0.001 are not shown. (b) Sizes of scaffolds >10 kbp, embedded is a histogram of RPKM values (the number of reads which map per kilobase of the exon model per million mapped reads), with expression at high salinity oriented outwards, and low salinity oriented inwards. (c) GC content in 1 kbp windows on a scale from 30% (yellow) to 60% (red). (d) Locations of certain groups of genes (red, energy production; blue, cell cycle; black, cell wall; green, membrane transporters). (e) Locations of repetitive sequences (grey, tandem repeats; blue, transposons; red tRNA). (f) Gene duplications and links, linking their locations determined by aligning the predicted proteins to the genome with Exonerate (cut-off: at least 50% of maximum score obtainable for each query). Blue, proteins that aligned with more than 200 amino acids; grey, proteins that aligned with 100–200 amino acids.
Figure 3
Figure 3
Genomic and proteomic comparison of Wallemia ichthyophaga (Wi) and Wallemia sebi (Ws). A. Dot-plot comparison of scaffolds longer than 200 kbp. The six-frame translations of scaffolds were aligned with Mummer 3.23. Homologous regions are plotted as dots. Scaffolds of each species are displayed ordered by decreasing size along x and y axes. Diagonal lines of dots in specific boxes represent syntenic regions. B. Shared and unique proteins of W. ichthyophaga and W. sebi, as determined by all-against-all blast (e-value cut-off, 10-6). C. Proportion of unique proteins that were matched to at least one Pfam family.
Figure 4
Figure 4
Extension and contraction of selected protein families. Proteins were classified into families according to the Pfam database, and the number of representatives in each family was analysed with the CAFE software. All families shown were significantly expanded (upward arrow) or contracted (downward arrow) in the proteome of Wallemia ichthyophaga. The numbers of representatives of each protein family in the contemporary species as well as the estimated ancestral states are shown in the trees. Statistically significant numbers for W. ichthyophaga, W. sebi and their last common ancestor are marked with an asterisk. The chronogram of analysed species was reconstructed on the basis of whole proteomes, and calibrated according to previously published calibration points in the fungal tree of life (scale unit, millions of years). A. Protein family of fungal hydrophobins (PF01185). B. Protein family of cation transporting (P-type) ATPases (PF00690). C. Major facilitator superfamily (MFS) and sugar transporters (PF07690, PF00083). D. Protein families of amino-acid permeases and transporters (AA uptake; PF13520, PF01490). E. Protein families of ATP binding cassette (ABC) transporters (PF01061, PF00664, PF00005, PF06422).
Figure 5
Figure 5
Analysis of significantly extended protein families. A. Gene tree of proteins with the Pfam domain PF00690 (P-type ATPase transmembrane transporters). The tree was constructed with the PhyML 3.0 software. Chi2-based approximate Likelihood-Ratio Test branch supports are shown for the major groups. Abbreviations of fungal species from which the proteins originate: Ab, Agaricus bisporus; An, Aspergillus nidulans; Ca, Candida albicans; Cc, Coprinopsis cinerea; Cn, Cryptococcus neoformans var. grubii; Mg, Malassezia globosa; Nc, Neurospora crassa; Pg, Puccinia graminis; Ro, Rhizopus oryzae; Rg, Rhodotorula graminis; Sc, Saccharomyces cerevisiae; Um, Ustilago maydis; Wi, Wallemia ichthyophaga; Ws, Wallemia sebi. The groups are labelled with the cations that are transported by the given group of ATPases, and the names of the S. cerevisiae proteins in that group (in brackets). B. Top: Proportion of acidic amino acids in hydrophobins from various fungal species. Box chart of quartiles shows the molar pecentages of acidic amino acids in individual hydrophobin proteins, with minima and maxima shown by the whiskers. Ustilago maydis contains only one hydrophobin of expected length. Bottom: The graphical representation of multiple sequence alignment of all hydrophobins from a given species shows the conservation of individual positions and amino acids. Black, acidic amino acids; light grey lined with black, cysteine residues; grey, all other residues.
Figure 6
Figure 6
Transcriptome of Wallemia ichthyophaga at 10% NaCl and 30% NaCl (w/v). A. Number of genes with increased expression at each salinity, compared to the other salinity. B. Number of novel transcripts predicted at each salinity. Gene models at least 200 bp away from known upstream or downstream genes were considered as novel transcripts. C. Proportion of proteins with increased expression at each salinity that were matched to at least one Pfam family. D. Number of genes with different alternative splicing modes at each salinity. Two intersecting circles show the number of genes with alternative splicing observed only at 10% NaCl, only at 30% NaCl, or at both (middle). E. Differences in expression of hydrophobins between the two salinities. Each bar represents a log2 ratio of expression at each salinity for an individual hydrophobin. All values above and below the dashed grey lines were considered as significantly differentially expressed.

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