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

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

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

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

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

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

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.

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

Number of virus and phage genomes sequenced

unclassifiedbacteriophages

ssDNA viruses

Satellites

ssRNA positive-strandviruses, no DNA stage

dsRNA viruses

Retro-transcribing viruses

unclassified viruses

negative-strand viruses

dsDNA viruses, no RNAstage

Deltavirus

Categories of phage genomes

Phage genomes vary in size from 4 kb up to 600 kb

Structural components of phage?

This animated GIF illustrates the process of a bacteriophage infecting a bacterial cell. (Coutesy of microbelibrary.org)

Phage of E.coli

Courtesy of microbelibrary.org

(Mehta et al. BMC Microbiology 2004)

The layout of a genome of lambdoid phage from non-pathogenic E. coli K-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)

Phage are the most abundant forms of life in the biosphere, and is estimated that there are

over 1031 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

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

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.)

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.

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

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

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

How do these regions compare in 4 E. coli O157:H7 phage genomes from 4 different isolated strains?

All 4 have stxA&BThese 2 have 3 tRNA

genes (in green)

These 2 do not have the 3 tRNA genes

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

Are 1 & 3 similar from the previous Salmonella phage

alignment similar to each other?

Only these two regions are similar

Comparison of 4 Yersinia pestis phage genomes

Which one of these is not like the others?

Alignment of 2 Shigella flexneri phage

Other than this collinear region, they are very dissimilar

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

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?

Phage clustered into groups based on Mauve alignments

Group #1

Group #2

Group #3

Group #4Stx 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

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

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

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)