1. 16S phylogeny of the Streptomycetaceae Alan Ward

2. Motivation Streptomyces taxonomy 16S Whole genome sequencing A bigger 16S phylogeny Where is the diversity The type strains Uncultured diversity Where are: The whole genomes Kitasatospora and Streptacidiphilus Marine actinomycetes Thermophiles The Streptomyces violaceusniger clade Who makes nigericin 16S phylogeny of the Streptomycetaceae Outline

3. Why? Hypothesis those claiming the same antibiotic in different species “are mistaken in species identification or the nature of the antibiotic” Krassilnikov 5 16S phylogeny of the Streptomycetaceae best producers of natural product antibiotics are members of the actinomycetes, and according to Hugenholz et al. (1998), approximately 30% or more of these have been discovered at one time or another. This has been the result of some fifty years worth of intense antibiotic screening programs conducted by pharmaceutical industry natural products scientists Strohl, 2004 For example, a survey of the literature linked the “species” names Streptomyces antibioticus, S. griseus and S. hygroscopicus, with the production of 13, 32, and 46 different structural types of natural products, respectively. Examples of compounds produced by strains characterized as S. hygroscopicus include rapamycin, spectinomycin, bialophos, hygromycin, and a variety of polyethers. Strohl 2004 To address this hypothesis, for Streptomyces, need to be able to address the relationship of strains to one another

5. 16S phylogeny of the Streptomycetaceae

6. Stackebrandt and Goebel (1994) Int J Syst Bacteriol 44, 846–849 - prokaryotes Stach et al. (2003) App Environ Microbiol 69, 6189-6200 - actinobacteria

8. Streptomyces sp. H668 Streptomyces sp. 371 Streptomyces sp. HGB0020 Streptomyces sp. HPH0547 Streptomyces sp. 118.1MFTsu1.1 Streptomyces sp. 142MFCol3.1 Streptomyces sp. 351MFTsu5.1 Streptomyces sp. AP10 JX101691 Streptomyces cinnamoneus Streptomyces thermoviolaceus Streptomyces sp. M17 Streptomyces sp. M43 Streptomyces sp. CNH099 Streptomyces sp. CNH189 Streptomyces sp. CNP082 Streptomyces sp. CNS654 Streptomyces sp. CNS615 Streptomyces sp. CNS335 Streptomyces sp. CNT371 Streptomyces sp. CNR698 Streptomyces sp. CNB091 Streptomyces sp. CNQ766 Streptomyces sp. CNS335 Streptomyces sp. CNS615 Streptomyces sp. CNT302 Streptomyces sp. CNT372 Streptomyces sp. CNY228 Streptomyces sp. CNY243 Streptomyces sp. PVA 94-07 Streptomyces sp. GBA 94-10 Streptomyces sp. CNS606 Streptomyces sp. CNT318 Streptomyces sp. TAA204 Streptomyces sp. CNQ329 Streptomyces sp. CNT360 Streptomyces sp. CNQ865 Streptomyces sp. TAA486 Streptomyces sp. CNH287 Streptomyces sp. TAA040 Streptomyces sp. CNQ-525 Streptomyces sp. GXT6 Streptomyces sp. PAMC26508 … 16S phylogeny of the Streptomycetaceae more Whole genome projects

9. Streptomyces acidiscabies 84-104Streptomyces scabiei 87.22 Streptomyces bingchenggensis BCW-1Streptomyces sp. S4 Streptomyces cattleya NRRL 8057Streptomyces sp. SA3_actF Streptomyces chartreusis NRRL 12338Streptomyces sp. SA3_actG Streptomyces chartreusis NRRL 3882Streptomyces sp. SirexAA-E Streptomyces clavuligerus ATCC 27064Streptomyces sp. TOR3209 Streptomyces coelicolor A3(2)Streptomyces sp. Tu6071 Streptomyces flavogriseus IAF45CD, ATCC 33331Streptomyces sp. W007 Streptomyces griseoaurantiacus M045Streptomyces sp. Wigar10 Streptomyces griseus griseus NBRC 13350Streptomyces sviceus ATCC 29083 Streptomyces griseus XyelbKG-1 1Streptomyces tsukubaensis NRRL 18488 Streptomyces hygroscopicus jinggangensis 5008Streptomyces turgidiscabies Car8 Streptomyces ipomoeae 91-03Streptomyces venezuelae ATCC 10712 Streptomyces lysosuperificus ATCC 31396Streptomyces violaceusniger Tu 4113 Streptomyces pristinaespiralis ATCC 25486Streptomyces viridosporus T7A, ATCC 39115 Streptomyces roseosporus NRRL 11379 16S phylogeny of the Streptomycetaceae Whole genome phylogeny Mohammad Tauqeer Alam, Maria Elena Merlo, Eriko Takano, Rainer Breitling (2010) Genome-based phylogenetic analysis of Streptomyces and its relatives. Molecular Phylogenetics and Evolution 54 763–772

10. Tree generation Downloaded 7,610 pre-aligned streptomycete 16S from RDP in Genbank FASTA format (in 2010) – remove 17 ambiguous sequences Edit labels to exclude invalid characters and consist of a unique numeric code, the species name, the strain name, the culture collection number and the Genbank sequence code Generate the pairwise alignment distance matrix Calculate the RDP alignment distance matrix and compare the pairwise and multiple alignment distances Repeat – Manually re-align and calculate the distance matrix – Calculate a tree (NJ-Jukes & Cantor initially then Maximum Likelihood) – Sort the sequences to tree order Until the total distance in the distance matrix stops decreasing 16S phylogeny of the Streptomycetaceae

11. Multiple alignment

12. 16S phylogeny of the Streptomycetaceae

13. Whole genome project s

14. 16S phylogeny of the Streptomycetaceae Higher order clusters? Kitasatospora Streptacidiphilus

15. 16S phylogeny of the Streptomycetaceae

16. 16S phylogeny of the Streptomycetaceae

17. 16S phylogeny of the Streptomycetaceae Streptomyces violaceusniger clade

18. 16S phylogeny of the Streptomycetaceae Streptomyces violaceusniger clade

19. 16S phylogeny of the Streptomycetaceae HPLC analysis of Streptomyces violaceusniger clade Aza He x Ela A Gel Pen B Number of strains producing each secondary metabolite in different production media. HPLC-DAD chromatograms showing major metabolites produced by members of the S. violaceusniger clade A, Streptomyces sp. DSM 4137 and B, WS-P18C9. Aza = azalomycin F, Ela = elaiophylin, Gel = geldanamycin, Hex = hexaene polyene and Pen = pentaene polyene.

21. 16S phylogeny of the Streptomycetaceae Streptomyces violaceusniger clade

22. Lankacidin Elaiophylin

23. 16S phylogeny of the Streptomycetaceae Blastp analysis of AntiSMASH for elaiophylin gene cluster

24. 16S phylogeny of the Streptomycetaceae AntiSMASH Streptomyces himastatinicus

26. 16S phylogeny of the Streptomycetaceae

27. Conclusions Motivation Streptomyces taxonomy 16S Whole genome sequencing A bigger 16S phylogeny Where is the diversity The type strains Uncultured diversity Where are: The whole genomes Kitasatospora and Streptacidiphilus Marine actinomycetes Thermophiles The Streptomyces violaceusniger clade Who makes nigericin

28. 16S phylogeny of the Streptomycetaceae Acknowledgements Stephen Haydock – for introducing us to Streptomyces DSM 4137 Porntipa Aiemsum-ang and Yashawant Kumar – PhD students at Newcastle Peter Fiedler Ern Lacy The software developersThe sequencers for Genbank/EMBLwhole genomes and gene clusters databases/blast/PubMed/links RDP 16S dataThe chemists for SeaViewmolecules and structures ClustalX Muscle MAFFT PhyML FastTree ESPRITCulture collections/microbiologists for Dendroscopemicroorganisms antiSMASH