Comparative Genomics of Aspergilli

1. Comparative Genomics of Aspergilli William Nierman TIGR

2. Electrophoretic Karyotyping 5 day run Af Sp Sc 5.7 5.0 4.6 1x 4.0 3.5 3.5 1.8 CHEF DRII 1.2% CGA, 1x TAE, 14C, 1.8 V/cm: 2200 s, 48 h; 2200-1800 s, 68 h sizes in Mb

3. A. fumigatus Chromosomes Size (MB) 1 4.891 4.834 4.018 3.933 3.922 3.779 2.021 1.789 2 3 ~35 copies rDNA 4 5 6 7 8 Centromeric area Telomere

4. Telomere repeat TTAGGG, 7-21 repeat units Subtelomeric regions- identical sequences for several kb, helicase pseudogenes, 7 secondary metabolite clusters, niche adaption role? (Mark Farman) Centromeres Uncloned in shotgun libraries; 36.2 - 55.9kb Flanked on each side by low complexity AT rich repeat region Chromosome 2 centromere 12 kb PCR product 75% AT, overall centromeric AT of 63%, 40kb. Centromeres and Telomeres

5. Annotation Pipeline Finished chromosome sequences Masked genomic sequence EST alignments Gene prediction Protein alignments Optimize Predictions Eukaryotic Genome Control (EGC) is the annotation pipeline responsible for processing genomic sequence

6. Full Length cDNAs (625) and 42 partials from 589 loci in 19 Aspergillus species 2,633 A. fumigatus ESTs from UK and Spanish collaborators Training Data Gene and splicing site predictions including Glimmer,Exonomy, Unveil, Phat and GeneSplicer were trained with following experimental data:

7. Optimize Predictions • Combiner combines gene model evidence from: • Gene prediction programs • Splice site prediction programs • Alignments from protein, cDNA and EST databases • Generates final gene model. All the genes were manual reviewed and the observed splits and merges were corrected.

8. Annotation Station Screenshot Scytalone dehydratase Yellowish-green 1,3,6,8-tetrahydroxynaphthalene reductase Polyketide synthetase Brown 1 Brown 2

9. Gene Summary Statistics

10. Most Common Domains in A. fumigatus

11. Synteny Map of A.fumigatus and A.nidulans

12. Synteny Map of A.fumigatus and A. oryzae

13. Synteny Map of A.fumigatus, A. nidulans, A. oryzae

14. Overview – Comparative Statistics The ortholog was computed by performing an all vs. all BlastP of the three proteomes with a cut-off of 1 x e-15 (no length requirement).  The mutual best hits were then organized into clusters based on shared protein nodes.

15. Status Count Total Sanger Genes analyzed 360 Same gene structure 137 Different gene structure 177 Sanger missing in TIGR annotation 37 Sanger matches multiple TIGR annotations 2 Sanger, TIGR annotations opposite strands 7 TIGR missing in Sanger annotation 12 TIGR matches multiple Sanger annotations 9 TIGR Autoannotation vs Sanger Curated Annotation

17. Using Ortholog Clusters to Identify Potential Annotation Problems

18. Using Ortholog Clusters to Identify Potential Annotation Problems Different exon number due to annotation discrepancy

19. In some cases, differences in exon number are real We need to be able to distinguish annotation inconsistencies from real, interesting phenomena

20. Apoptosis-like process detected in S. cerevisiae, S. pombe, and Aspergilli. Fungal genomes lack metazoan upstream machinery. Metacaspase-dependent phenotype observed in A. fumigatus and A. nidulans. Analysis by Goeff Robson Apoptosis in Fungi

21. Apoptosis in Fungi

22. Aspergillus fumigatus Secondary Metabolites Heterogeneous group of low molecular weight products. Toxic, antibiotic, and immunosuppressant activities. – fumagillin, gliotoxin (apoptosis and phagocyte dysfunction), fumitremorgin, verruculogen, fumigaclavine, helvolic acid, phthioc acid (granulomas when injected into mice) and sphingofungins Virulence properties may be augmented by the A. fumigatus numerous secondary metabolites.

23. Gene type A. oryzae A. fumigatus A. nidulans PKS 30 14 27 NRPS 18 14 14 FAS 5 1 6 Sesquiterpene cyclase 1 (1) (1) DMATS 2 7 2 Secondary Metabolite Genes Analysis by G. Turner, N. Keller, Dr. Kitamoto, and R. Kulkarni

24. Serine Phenylalanine 2 module NRPS? Gliotoxin Terpene Sesquiterpene cyclase Fumagillin Tryptophan DMAT synthetase (X2) Fumigaclavines Tryptophan Proline NRPS? DMAT synthetase Fumitremorgens

25. Gene type A. oryzae A. fumigatus A. nidulans PKS2 30 14 27 NRPS 18 14 14 FAS 5 1 6 Sesquiterpene cyclase 1 (1) (1) DMATS 2 7 2 Five 2-module NRPS

26. Few true orthologues across the genus Aspergillus. Each species has its own repertoire. Gene/product relationship requires functional analysis in most cases Indole alkaloid pathway in A. fumigatus only. Closely related to Claviceps purpurea ergotamine pathway Penicillin and aflatoxin pathways are absent. A hybrid PKS/monomodular NRPS seems to be present in several fungi. A. fumigatus Secondary Metabolite Genes

27. Identify A.fumigatus specific genes A.fumigatus genes (9746) All vs. all BlastP of the AFU1,ANA1, AOAN proteomes cut-off E value: 1 x e-15, filtering the results for mutual best hits between genomes. A. fumigatus singletons (2075) BLASTP vs ANA1 and AOA1 proteomes A. fumigatus singletons E-value > e-10 (1081) Extend 50bp on both ends of the gene in the genome, Tblastx the genomic seq of the gene vs ana and aoa genomic seq A. fumigatus specific gene candidates E-value > e-50 (1011) BLASTP vs ANA1 and AOA1 proteomes e-5>E-value>e-10 E-value > e-5 (203) (808) Extend 50bp on both ends of the gene in the genome, Tblastx the genomic seq of the gene vs ana and aoa genomic seq e-50<E-value < e-10 e-5>E-value>e-10 E-value > e-5 (552) (181) (75)

28. Vast majority are hypothetical Includes Several transcriptional regulators A chaperonin An hsp 70 related protein Aspergillus fumigatus Unique Genes

29. 19th century poisonings associated with green pigments. 1892 B. Gosio, certain fungi could metabolize arsenic pigments producing toxic trimethylarsine (Gosio gas). Screen in the 1930s (Thom & Raper) found A. fumigatus to be an arsenic fungus. Napoleon, imperial colors green and gold, copper arsenite (Jones 1982). Analysis of history and genome by J. Bennett, N. Hall, J. Wortman, C. Lu. Arsenic Fungi

30. Arsenite efflux pump Arsenite translocating ATPase Two possibly duplicated clusters arsC – arsenate reductase (A. fumigatus unique) arsB – arsenite symporter arsH Methyltransferase A. fumigatus Arsenate Genes

31. Chromosome 5 Chromosome 1

32. arsB Methytrasferase arsH arsC arsB Methyltransferase arsH arsC

33. Good homology to a the full length of the Streptomyces griseus protein. Secretion signal peptide may direct for cell wall. Teichoic acids demonstrated to be a virulence factor for Staphylococcus aureus. No intervening sequences in gene. A. Fumigatus Teichoic Acid Biosynthesis Protein Analysis by Neil Hall

34. More highly expressed at 48oC More highly expressed at 37oC A. Fumigatus Thermotolerance

35. Relatively few genes altered Some HSPs transiently or stably induced (weakly) and repressed at 37oC. HSPs induced throughout 180 min 48oC period Transposases induced at 48oC (Mariner 4). Stress related genes up regulated at 48oC. Metabolic proteins down regulated at 48oC A. fumigatus Thermotolerance “This fungus likes it hot.” J. Bennett

36. Microarray Detection of Clusters

37. Aspergillus fumigatus AF293 Project Participants The University of Manchester, UK The Wellcome Trust Sanger Centre, UK The Institute for Genomic Research, USA The University of Salamaca, Spain Complutense University, Spain Centro de Investigaciones Biológicas, Spain

38. Aspergillus fumigatus AF293 Joan Bennett Matt Berriman Jean Paul Latge Paul Dyer Paul Bowyer Neil Hall David Denning Michael Anderson Arnab Pain Goeff Robson Javier Arroyo Goeff Turner David Archer Aspergillus nidulans – James Galagan Aspergillus oryzae – Masayuki Machida

39. TIGR Sequencing and Closure Tamara Feldblyum Hoda Khouri Annotation Jennifer Wortman Jiaqi Huang Resham Kulkarni Natalie Fedorova Charles Lu Lab Group Heenam Kim Dan Chen Claire Fraser NIAID and Dennis Dixon