Black Yeasts Conference “The black fungi around us – harm and benefit”

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1 Black Yeasts Conference “The black fungi around us – harm and benefit”
Guangzhou (China), 29 November - 1 December 2013 Physiological analysis and karyotyping of the two rock-inhabiting black yeasts Knufia petricola A95 and Coniosporium apollinis Corrado Nai & Anna A. Gorbushina Free University of Berlin &  BAM Federal Institute for Materials Research and Testing Department “Materials & Environment”

2 Knufia petricola (basionym Sarcinomyces petricola) A95 A model rock-inhabiting fungus
Phylogenetic position described (Chaetothyriales, ancestral clade) Typical microcolonial traits Stress tolerance (pigments) Melanised Meristematically growing Oligotrophic Typical origin: marble in Athens Substrate interaction Acceptable growth rate Scattered colony development Genome sequence analysis is underway (454 and Illumina)

3 Knufia petricola (basionym Sarcinomyces petricola) A95 A model rock-inhabiting fungus
Broad physiological profile using Biolog Phenotype MicroArray (PM) plates ̴1‘000 different conditions (salt stress and pH optimum, growth factor requirement, nutrient utilization) No C source Different salt concentrations Different C sources (other wells) C metabolism No supplements Salt stress Different vitamins / growth factors (other wells) Vitamin requirement N metabolism Bochner (2003). New technologies to assess genotype-phenotype relationships. Nature Review Genetics 4: Nai C et al. (2013). Nutritional physiology of a rock-inhabiting, model microcolonial fungus from an ancestral lineage of the Chaetothyriales (Ascomycetes). Fungal Genetics and Biology 56:54-66.

4 Knufia petricola (basionym Sarcinomyces petricola) A95 A model rock-inhabiting fungus
Broad physiological profile using Biolog Phenotype MicroArray (PM) plates ̴1‘000 different conditions (salt stress and pH optimum, growth factor requirement, nutrient utilization) Halotolerant, pH optimum ̴ pH 5 Prototroph, but stimulated by thiamine Selective utilization of C source ( ̴33 %), no uptake of metabolic intermediates, growth on monoaromatic compounds (p-hydroxybenzoic acid) Broad utilization of N ( ̴75 %) and P compounds ( ̴50 %) Bochner (2003). New technologies to assess genotype-phenotype relationships. Nature Review Genetics 4: Nai C et al. (2013). Nutritional physiology of a rock-inhabiting, model microcolonial fungus from an ancestral lineage of the Chaetothyriales (Ascomycetes). Fungal Genetics and Biology 56:54-66.

5 Coniosporium apollinis vs. Knufia petricola A95
Same ecology Same origin Similar morphology Both sequenced (Broad) Different phylogeny Knufia petricola ( ) © C. Gueidan

6 K .petricola A95 CBS C. apollinis CBS © C. Gueidan

7 C. apollinis vs. K. petricola A95
Biolog analyses (as in Nai C et al. 2013) ca. 500 conditions (salt tolerance, pH optimum, C and N metabolism) Karyotyping (number of chromosomes) by pulsed field gel electrophoresis

8 Biolog data of C. apollinis General growth requirements (I) – salt stress (PM9)
C. apollinis is halotolerant (but less than A95)

9 Biolog data of C. apollinis General growth requirements (II) – pH plate (PM10)
Broader pH range (pH 4.5 to 9) Shifted toward basic pHs pH optimum at around pH 5-6

10 Biolog data of C. apollinis Carbon source utilization
C. apollinis is even more selective than A95 (only ̴ 16 % C sources used) Major differences with A95: Pentose catabolic pathway (growth on the intermediates adonitol, L-arabitol, xylitol) Galactose pathway (no growth D-galactose and thus seemingly not active) Starch and sucrose metabolism (more active) Aromatic pathway (none of the compounds used) Good growth on sugar alcohols Growth on D-sorbitol, D-melibiose and stachyose is not supportive of activity of galactose pathway since D-sorbitol is also in the glycolytic pathway and the two oligosaccharides are composed of glucose which is probably responsible for growth.

11 Biolog data of C. apollinis Nitrogen source utilization
Again, C. apollinis is more selective than A95 ( ̴ 37 % N compounds used) D-amino acids and primary amines (except tyramine): all negative

12 Biolog data of C. apollinis Summary
C. apollinis K. petricola A95 Osmotolerance (up to 4 % NaCl) (up to 8-10 % NaCl) pH optimum pH 5-6 (growth: pH 4.5-9) pH 5 (growth: pH 3.5-7) C metabolism Very selective (ca. 15 % used) Selective (ca. 33 % used) Different pathways active/inactive (metabolic intermediates, polyols used by C. apollinis) N metabolism Selective (ca. 37 % used) Rather unselective (ca. 50 % used) D-aa and primary amines not used

13 Karyotyping (PFGE) - protocol
Grow cells (MEB) into early stationary phase (low melanization) Resuspend in protoplast buffer and treat with 10 mg/mL “Lytic Enzymes from Trichoderma harzianum“ (LETh) (Sigma) – mixture of β-glucanase, cellulase, protease and chitinase – for 1-2 d at 27 °C Isolate protoplasts by gradient centrifugation (0.8 M sucrose & 1.5 % w/v Ficoll) Count protoplasts, embed in low-melting point agarose Treat plugs with -mercaptoethanol (disrupt cell membranes) and Proteinase K (digest proteins) (additional protoplasting step in situ with LETh possible) Run PFGE (ROFE) 4‘000 x g in sucrose-ficoll solution

14 Karyotyping: First results with K. petricola A95

15 Karyotyping: First results with K. petricola A95
7-8 choromosomes Genome size estimation: 29.4 Mb (Broad Institute: Mb)

16 Karyotyping: First results with C. apollinis
12 choromosomes Genome size estimation: 14.3 Mb (Broad Institute: Mb !)

17 Karyotyping: First results with C. apollinis
12 choromosomes Genome size estimation: 14.3 Mb (Broad Institute: Mb !) …no bigger bands ! Co-migrating chromosomes? Underestimation of genome size by PFGE?

18 Acknowledgements Serap Avci Ronald Banasiak William Broughton Nicole Knabe Pedro Martín Sánchez Steffi Noack Franz Seiffert Jörg Toepel Leoni Lang Marco Tosi Helen Wong