1. PHANTOME: Phage Annotation Tools And Methods Rob Edwards San Diego State University Argonne National Laboratory

2. Tree of Life

4. Outline ● Phages versus prophages ● Better phage annotations ● How phages die

5. Rossmann Lab, Purdue http://bilbo.bio.purdue.edu/~viruswww/Rossmann_home/

6. Phages and Prophages

7. Finding Prophages Similarity to other phages

8. Finding Prophages Use phage integration to delimit the phage in the genome attP attB attL attR

9. Finding Prophages Use phage integration to delimit the phage in the genome attP attB attL attR Core duplications

10. Core Duplications Exact nucleotide repeats flank the phage Phage λ: 15 bp (tyrosine recombinase) Shortest duplication: 3 bp Streptomyces φC31 (serine recombinase) Can we find other core duplications surrounding prophage?

11. Phantometrics

12. Where Do Phages Insert In The Genome?

13. t RNA genes have 2° structure http://www.web-books.com

14. Frequency of Insertions

15. Core sites

16. How long are core duplications?

17. Core Duplication Lengths 60 50 40 30 20 10 0 1-10 11-20 21-30 41-50 31-4051-60 61-70 71-80 81-90 91-100 101-200 >200 Core duplication length (nt) Number of insertions Median size: 21 nt

18. 16 14 12 8 4 0 1-10 11-20 21-30 41-50 31-4051-60 61-70 71-80 81-90 91-100 101-200 >200 Core duplication length (nt) Number of insertions Median size: 32 nt Core Duplication Lengths ( t RNA insertions)

19. t RNA Genes Must Be Reconstituted attP attB attL attR tRNA gene 'tRNA gene

20. Outline ● Phages versus prophages ● Better phage annotations ● How phages die

21. Phage Annotations

22. www.phantome.org AIMS: ● Consistency ● Biological relevance ● Rapid propagation ● Compatibility

23. Capsid proteins Tail proteins Replic- ation Packaging: portals and terminases Lysis Lysogenic Conversion Lysogeny: Integration/ Excision Trans- cription Regln Functional modulesStructural modules Information transfer Phage-host interactions Neck HeadTail Baseplate Tail fibers Packaging Most Common Phage Modules

24. Conserved Genes Across Many Genomes Enterobacter sp 638 E. coli APEC prophage S. enterica sub. enterica K. pneumoniae MGH 78578 Yersinia frederiksenii Ralstonia solanacearum Proteus mirabilis Xanthomonas axonopodis Haemophilus influenzae 1: Terminase, endonuclease subunit, 2: Terminase, ATPase subunit, 3: Head completion/stabilization protein, 4: Capsid protein, 5: Capsid scaffolding protein, 6: Tail protein, 7: Portal protein

25. Conserved – But Different! Toxin AttR Lysin Similar chromosomal insertion region Paratox Unrelated toxins: nucleases or superantigens Phage lysins: two different types of lysins 2: Hyaluronidase

26. How do (pro)phages die?

27. Two Insertions At Same Place CPP-933H tRNA thrW Truncated tRNA thrW

28. Two Insertions At Same Place CPP-933I Truncated tRNA thrW tRNA thrW CPP-933H

29. Mismatches Between attL and attR Secondary sites may not be exact sequence match to attP Core does not have same sequences on both sides Imperfect repeats: – 12% of all insertions – 22% of tRNA insertions

30. IS elements per phage 30 20 10 0 68 14162412 Number of phage Number of IS elements per phage All phages Defective phages Suspected viable phages Viable phages

31. IS Elements: Phage or Genome? Significantly different using χ 2 at P<0.01 and 1 d.f.

32. 60 50 40 30 20 10 0 1-10 11-20 21-30 41-50 31-4051-60 61-70 71-80 81-90 91-100 101-200 >200 Number of insertions Core Duplication Lengths Core duplication length (nt) Phage λ-like Serine recombinases Insertions in tRNAs IS elements

33. Summary Not all prophages insert at tRNAs Core duplications flank almost 50% of prophage Insertions at tRNAs are longer than insertions elsewhere – Requirement for function? – Ancestral? Prophages are killed by IS elements – More frequently than other regions of genome

34. Acknowledgements

35. How do prophages die? Two insertions at same place Mismatches Transposons and IS elements