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

Tree of Life

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

Rossmann Lab, Purdue

Phages and Prophages

Finding Prophages Similarity to other phages

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

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

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?

Phantometrics

Where Do Phages Insert In The Genome?

t RNA genes have 2° structure

Frequency of Insertions

Core sites

How long are core duplications?

Core Duplication Lengths >200 Core duplication length (nt) Number of insertions Median size: 21 nt

>200 Core duplication length (nt) Number of insertions Median size: 32 nt Core Duplication Lengths ( t RNA insertions)

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

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

Phage Annotations

AIMS: ● Consistency ● Biological relevance ● Rapid propagation ● Compatibility

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

Conserved Genes Across Many Genomes Enterobacter sp 638 E. coli APEC prophage S. enterica sub. enterica K. pneumoniae MGH 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

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

How do (pro)phages die?

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

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

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

IS elements per phage Number of phage Number of IS elements per phage All phages Defective phages Suspected viable phages Viable phages

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

>200 Number of insertions Core Duplication Lengths Core duplication length (nt) Phage λ-like Serine recombinases Insertions in tRNAs IS elements

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

Acknowledgements

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