Transcript Understanding phage, the viruses that infect

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
Courtesy of pbs.org
•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.
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 fX174 genome (Nobel prize 1980). This
was the first complete genome sequence of any
organism to be determined.
-Bacteriophage fX174, 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
Credit: Robert Duda, University of Pittsburgh
This method allowed
sequencing projects to
proceed at a much faster
rate thus expanding the
scope of realistic
sequencing venture.
(Sanger et al. J. Mol. Biol. 1982)
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)
Categories of phage genomes
Number of virus and phage genomes sequenced
unclassified
bacteriophages
26 unclassified
bacteriophages
ssDNA viruses
324 ssDNA viruses
Satellites
99 Satellites
518 ssRNA positive-strand
viruses, no DNA stage
99 dsRNA viruses
ssRNA positive-strand
viruses, no DNA stage
dsRNA viruses
Retro-transcribing viruses
92 Retro-transcribing viruses
unclassified viruses
7 unclassified viruses
104 negative-strand viruses
525 dsDNA viruses, no RNA
stage
negative-strand viruses
dsDNA viruses, no RNA
stage
Deltavirus
1 Deltavirus
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
The layout of a genome of lambdoid phage from nonpathogenic E. coli K-12
(Mehta et al. BMC Microbiology 2004)
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
Courtesy of nasa.gov
-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
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
StxB2
(subunitA) (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
933EDL strain from an
outbreak in ground beef (top)
Sakai strain from an
outbreak of radish
sprouts (bottom)
Courtesy of usbg.gov
Courtesy of cfsan.fda.gov
Divergent
region
Otherwise highly conserved
How do these regions compare in 4 E. coli O157:H7 phage
genomes from 4 different isolated strains?
These 2 have 3 tRNA
genes (in green)
These 2 do
not have the
3 tRNA
genes
All 4 have
stxA&B
How about phage from another human pathogen Salmonella spp.?
Phage 2,4,&5
1
are similar
with the green
and red
2
regions,
whereas 1 & 3
3
are not similar
to this cluster
4
Are 1 & 3
similar to
each other?
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?
Salmonella
ST104 phage
In some cases yes
Shigella Sf6
phage
Non-pathogenic
E. coli HK620
phage
2 Shigella flexneri phage
5 Salmonella spp. phage
How do 19 different
phage from the family
Enterobacteriaceae
align?
4 Yersinia pestis phage
3 E. coli O157:H7 phage
5 non-pathogenic E. coli phage
Phage clustered into groups
based on Mauve alignments
Shigella flexneri SfV &
Salmonella spp. ST64B
Shigella flexneri Sf6;
Salmonella spp. ST64T,
ST104, and P22; and E.
coli HK260
Y. pestis phage
Group #1
Group #2
Group #3
Stx phage from Group
E. coli O157:H7
#4
Phage are even more diverse in relationship
than bacteria
5 phage from
the same E.
coli host, are
grouped in
two different
taxonomic
groups
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)