1. Understanding phage, the viruses that infect microorganisms, via genome alignments

2. History of phage? Bacteriophage def:(bacteria and the greek work phagein, “to eat”) In 1896, Ernest Hanbury Hankin reported that something in the waters of the Ganges and Jumna rivers in India had marked antibacterial action against cholera and could pass through a very fine porcelain filter In 1915, British bacteriologist Frederick Twort, superintendent of the Brown Institution of London, discovered a small agent that infects and kills bacteria. He considered the agent either 1) a stage in the life cycle of the bacteria, 2) an enzyme produced by the bacteria itself or 3) a virus that grows on and destroys the bacteria. Bathers in the Ganges River at Benares, India Courtesy of nlm.gov

3. History continued Independently, French-Canadian microbiologist Félix d'Hérelle, working at the Pasteur Institute in Paris, announced on September 3, 1917 that he discovered "an invisible, antagonistic microbe of the dysentery bacillus". For d’Herelle, there was no question as to the nature of his discovery: "In a flash I had understood: what caused my clear spots was in fact an invisible microbe... a virus parasitic on bacteria." D'Herelle called the virus bacteriophage or bacteria-eater (from the Greek phago meaning to eat). He also recorded a dramatic account of a man suffering from dysentery that the bacteriophages restored to good health. Courtesy of pbs.org

4. One of the causative agents of dysentery was called dysentery bacillus, which was later identified as Shigella spp. We have the genomes of four Shigella species housed in ERIC (strains in parentheses). Shigella flexneri (strains 2457T, 301, and 8401) Shigella sonnei (046 and 53G) Shigella dysenteriae (197, 1012, and M131649) Shigella boydii (BS512 and 227) Courtesy of cfsan.fda.gov

5. History of DNA sequencing and genome research involved phage -Frederick Sanger and colleagues determine the complete sequence of all 5,375 nucleotides of the bacteriophage  X174 genome (Nobel prize 1980). This was the first complete genome sequence of any organism to be determined. -Bacteriophage  X174, was the first genome to be sequenced, a viral genome with only 5,368 base pairs (bp) (Sanger et al., Nature 1977) Courtesy of nlm.gov

6. Sanger first used "shotgun" sequencing five years later to complete the bacteriophage lambda sequence that was significantly larger, 48,502 bp (Sanger et al. J. Mol. Biol. 1982) This method allowed sequencing projects to proceed at a much faster rate thus expanding the scope of realistic sequencing venture. Credit: Robert Duda, University of Pittsburgh

7. Sequenced viral genomes that followed had progressively larger genome sizes. -229 kb genome of cytomegalovirus (CMV) (genus of herpes viruses) -the 192 kb genome of vaccinia ~250 genes (poxvirus family) used as a tool for gene therapy -186 kb genome of smallpox.

8. There are now many types of phage and virus genomes sequenced (1,795 as of 5/2007) 26 unclassified bacteriophages 324 ssDNA viruses 99 Satellites 518 ssRNA positive-strand viruses, no DNA stage 99 dsRNA viruses 92 Retro-transcribing viruses 7 unclassified viruses 104 negative-strand viruses 525 dsDNA viruses, no RNA stage 1 Deltavirus Categories of phage genomes Phage genomes vary in size from 4 kb up to 600 kb

9. Structural components of phage? This animated GIF illustrates the process of a bacteriophage infecting a bacterial cell. (Coutesy of microbelibrary.org)

10. Phage of E.coli Courtesy of microbelibrary.org

11. (Mehta et al. BMC Microbiology 2004) The layout of a genome of lambdoid phage from non- pathogenic E. coli K-12

12. Relationships among phage and prophage solid lines representing sequence similarities and the dotted lines corresponding to commonalities of gene organization or gene function. Closely related phages are shown in boxes, and bacterial hosts are shown at the perimeter of the web (Hendrix et al. 1999)

13. Phage are the most abundant forms of life in the biosphere, and is estimated that there are over 10 31 tailed bacteriophages on Earth -phage genome architecture may have occurred due to extensive horizontal gene transfer over the last 3 billion years ( As more phage genomes are sequenced, we will start to understand the immense diversity of phage Courtesy of ncbi.nlm.nih.gov Courtesy of nasa.gov

14. Do phage cause human disease? In some cases they contribute virulence factors of pathogenic bacteria -one example is the human shiga toxin (Stx2) which is encoded within the phage sequence of Escherichia coli O157:H7. The first E. coli O157:H7 genome sequenced was from strain 933 EDL (Perna et al. 2001) Stx2 toxin encoded in the phage genome from the bacterial genome phage genome StxA2 (subunitA) StxB2 (subunitB) Bacterial genome

15. Mauve Multiple Genome Aligner Able to identify and align collinear regions of multiple genomes even in the presence of rearrangements Find and extend seed matches Group into locally collinear blocks Align intervening regions (Darling et al. Genome Res. 2004 Jul;14(7):1394-403.)

16. Genomic Context: a larger scale Mauve: Multiple Alignment of Conserved Genomic Sequence With Rearrangements (Darling et al. Genome Research 2004) Mauve has been developed with the idea that a multiple genome aligner should require only modest computational resources. It employs algorithmic techniques that scale well in the amount of sequence being aligned. For example, a pair of Y. pestis genomes can be aligned in under a minute, while a group of 9 divergent Enterobacterial genomes can be aligned in a few hours.

17. Comparing phage genomes using Mauve -Phage genomes can be aligned using Mauve in a matter of minutes. -applicable as a teaching tool to decipher the mosaicism of phage genomes. -comparative studies of 30 mycobacteriophage genomes reveal new insights into the diverse architecture and insight about gene exchange (Hatfull et al. PLoS genetics et al. 2006) -How diverse are enterobacteriophage? (the following series of slides are Mauve alignments of phage isolated from E. coli, Salmonella spp., Yersinia spp., and Shigella spp.) all alignments are also provided for further inquiry

18. How do lambdoid phage from a non-pathogen (E. coli K-12) compare with the phage Stx2 from a pathogen (E. coli O157:H7)? Some regions are similar Some are highly divergent and different

19. Lets compare the 2 E. coli O157:H7 Stx-phage from the Sakai (RIMD) and EDL933 strains Divergent region Otherwise highly conserved 933EDL strain from an outbreak in ground beef (top) Sakai strain from an outbreak of radish sprouts (bottom) Courtesy of cfsan.fda.gov Courtesy of usbg.gov

20. How do these regions compare in 4 E. coli O157:H7 phage genomes from 4 different isolated strains? All 4 have stxA&B These 2 have 3 tRNA genes (in green) These 2 do not have the 3 tRNA genes

21. How about phage from another human pathogen Salmonella spp.? Phage 2,4,&5 are similar with the green and red regions, whereas 1 & 3 are not similar to this cluster Are 1 & 3 similar to each other? 1 2 3 4 5

22. Are 1 & 3 similar from the previous Salmonella phage alignment similar to each other? Only these two regions are similar

23. Comparison of 4 Yersinia pestis phage genomes Which one of these is not like the others?

24. Alignment of 2 Shigella flexneri phage Other than this collinear region, they are very dissimilar

25. Are phage from different genera of bacteria more similar? In some cases yes Salmonella ST104 phage Shigella Sf6 phage Non-pathogenic E. coli HK620 phage

26. 2 Shigella flexneri phage 5 Salmonella spp. phage 4 Yersinia pestis phage 3 E. coli O157:H7 phage 5 non-pathogenic E. coli phage How do 19 different phage from the family Enterobacteriaceae align?

27. Phage clustered into groups based on Mauve alignments Group #1 Group #2 Group #3 Group #4 Stx phage from E. coli O157:H7 Y. pestis phage Shigella flexneri SfV & Salmonella spp. ST64B Shigella flexneri Sf6; Salmonella spp. ST64T, ST104, and P22; and E. coli HK260

28. 5 phage from the same E. coli host, are grouped in two different taxonomic groups Phage are even more diverse in relationship than bacteria Courtesy of Pittsburgh Bacteriophage Institute

29. Taxonomy of phage -Unlike microorganisms, conserved genes to determine taxonomy such as the 16S rRNA gene don’t exist in phage -it has been proposed that phage integrase gene can be used to infer phylogeny, and has been used to cluster 31 enteric phage Courtesy of nsf.gov

30. Taxonomy of enteric phage -tyrosine recombinase family was used since members of this family have been isolated from archaea, bacteria and their phages, from a mitochondrion and from yeast (Balding et al. 2005)