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Henry Potosnak
Period 3
March 30, 2009
Independent Study Report
Bacteriophage Discovery and Characterization Research Project
I.
Introduction
Purpose
The purpose of this investigation is to attempt to discover a never- before-classified
bacteriophage. The research involves finding a novel bacteriophage and then characterizing it.
The techniques employed to characterize the identified phage include electron microscopy;
sequencing, annotating, and analyzing the genome of the phage; and comparing the genome of
your phage to other sequenced phage genomes.
Background
The goal of this study is to discover a new species of bacteriophage. Bacteriophages are
viruses that infect, feed off of, and destroy bacteria. They are much smaller than bacteria. A
bacteriophage’s size is generally between 20 and 200 nanometers. Bacteriophages typically
consist of an outer protein1 encasing genetic material. Their four genetic structure
configurations are: ssRNA, dsRNA, ssDNA, or dsDNA. The “ss” or “ds” indicates whether the
DNA/RNA is single-stranded or double-stranded.
Bacteriophages have some of the largest populations of organisms on Earth. It is
thought that they are the most amply dispensed and assorted organisms in our biosphere.
Bacteriophages are practically universal. They can survive in almost any environment. If
bacteria exist in a location, it is quite likely that bacteriophages exist there as well. The
preeminent place to find bacteriophages (and other viruses) is the ocean. The population of
bacteriophages in sea water can be so dense that some microbial mats have been found to
contain up to 9x108 virions per milliliter. It is estimated that up to 70% of marine bacteria may
be infected by bacteriophages.2
The group of scientists who classify bacteriophages is referred to as the International
Committee on Taxonomy of Viruses (ICTV). The ICTV has found a few orders to classify
bacteriophages, but most bacteriophages belong to just one order, Caudovirales. Caudovirales
are the dsDNA tailed bacteriophages. 95% of all the recorded bacteriophage findings belong to
the Caudovirales.3 It is possible that Caudovirales make up the majority of bacteriophages on
our planet. Following is a list of the families of the Caudorvirales’ order:
ICTV Classification of Bacteriophages
Order
Family
Morphology
Nucleic acid
Myoviridae
Non-enveloped, contractile tail
Linear dsDNA
Siphoviridae
Non-enveloped, long non-contractile tail
Linear dsDNA
Podoviridae
Non-enveloped, short noncontractile tail
Linear dsDNA
Tectiviridae
Non-enveloped, isometric
Linear dsDNA
Corticoviridae
Non-enveloped, isometric
Circular dsDNA
Lipothrixviridae
Enveloped, rod-shaped
Linear dsDNA
Plasmaviridae
Enveloped, pleomorphic
Circular dsDNA
Rudiviridae
Non-enveloped, rod-shaped
Linear dsDNA
Fuselloviridae
Non-enveloped, lemon-shaped
Circular dsDNA
Inoviridae
Non-enveloped, filamentous
Circular ssDNA
Microviridae
Non-enveloped, isometric
Circular ssDNA
Leviviridae
Non-enveloped, isometric
Linear ssRNA
Cystoviridae
Enveloped, spherical
Segmented dsRNA
Caudovirales
Bacteriophages were discovered by a French-Canadian microbiologist Félix d'Hérelle.
d'Hérelle was working at the Pasteur Institute in Paris when he announced on September 3,
1917 that he had found “an invisible antagonistic microbe of the dysentery bacillus.” He said,
“in a flash I had understood: what caused my clear spots was in fact an invisible microbe … a
virus parasitic on bacteria.” He named it a bacteriophage (from the Greek word phagein, which
means: to eat4).
The two cycles used by bacteriophages for replication are: 1) a lytic cycle, and 2) a
lysogenic cycle.5 Most bacteriophages can only use one of these cycles. A few can use both. In
the lytic cycle, a bacterial cell is burst agape (lysed) and eradicated after immediate cloning of
the virion. After the bacterial cell is destroyed, the new bacteriophages can find another soonto-be-destroyed host. The less violent lysogenic cycle does not end in immediate lysing of the
host cell. The bacteriophages that follow this cycle are known as temperate phages. In this
cycle, bacteriophages integrate their viral genome with the host’s DNA and replicate with it.6
Sometimes, it may establish itself as a plasmid. The bacteriophage keeps the cell living and
reproducing, because the virus is reproduced in every replicated cell. In the lysogenic cycle,
bacteriophages do not destroy the host. They become long-term occupants. The bacteriophage
will remain dormant until the host’s health weakens, which may be due to loss of nutrients.
Then, it activates, begins the reproductive cycle, and eventually ends in the lysing of the host
cell.
Bacteriophages use several different adaptations to attach themselves to their bacterial
host. The known adaptations are:




Lipopolysaccharides
Teichoic acids
Proteins
Flagella.
Since different bacteriophages have specific adaptations to attach themselves to bacteria, each
bacteriophage can attach to certain bacteria. The bacteria need the matching receptors to the
bacteriophages’ adaptations. Bacteriophage virions cannot move independently, so they must
rely on random encounters with matching receptors.
After connecting with the matching receptor, the bacteriophages begin their assault on
the bacteria. More complex bacteriophages use a motion that can be compared to that of a
syringe to inject their genetic material into the bacteria cell. Other bacteriophages use their tail
fibers to move closer to the bacteria cell’s surface. Next, the bacteriophages attach themselves
to the surface completely, contract their tail (which could be possible due to the ATP in the tail),
and inject their genetic material through the bacteria’s cell membrane.
It only takes a few minutes to override and replace the bacteria’s RNA with the virus’s
mRNA. The host’s ability to synthesize proteins and nucleic acids gets confused. It now must
construct viral crops instead. The viral products and the helper proteins are used to create new
virions within the cell. Some of the proteins are used in the cell for lysis.
Once the bacterial cell has been used up, the bacteriophages will exit the cell via lysis,
extrusion, or rarely by budding. Bacteriophages with tails use an enzyme known as endolysin.
Endolysin breaks down and destroys the cell wall peptidoglycan. Another type of
bacteriophage, the filamentous phage, forces the host cell to constantly excrete new virus
particles. Budding is affiliated with specific Mycoplasma phages.
Bacteriophages can and are being used to stop and prevent bacteria from infecting
humans. In August, 2006, the United States Food and Drug Administration (FDA) approved the
use of bacteriophages on cheese to eliminate the Listeria monocytogenes bacteria, giving them
the status of GRAS (Generally Recognized as Safe). In July 2007, the very same bacteriophages
were approved for use on every food product. Bacteriophage are even being used in hospitals
as a preventative treatment for medical devices. New technology now allows for
bacteriophages to be applied to dry surfaces. There is even research into their potential to
serve as an offense to bio-terror events.
II.
Procedure
Attachment 1 provides the detailed procedure for the experiments required for this
investigation. I conducted these experiments using microbiology and molecular biology
techniques as a volunteer participant in the Phagehunting independent research program in the
Phage hunter lab of Dr. Graham Hatfull at the University of Pittsburgh’s Department of
Biological Sciences and under the mentorship of Nicolle Gill. The project is funded by a Howard
Hughes Medical Institute Professorship grant.
III.
Results
Attachment 2 provides the results recorded during the weekly research sessions. The
notebook pages contain details of each step performed as well the results and observations
noted.
IV.
Conclusions
Sample 00784 contains a phage. Sample 00784 was plated out from 10-1 to 10-10. A
plaque was picked from plate 10-5. That pick was plated, and showed five different types of
plaques. Plaque one was a larger, more opaque plaque. Plaque one was the plaque picked for
purification. After four rounds of purification, only plaque one remained. Plaque one was
identified as a bacteriophage.
The following steps will be done in future lab sessions. The lysate will be filtered through
a .22 micro liter filter. The next step will be to determine the concentration, or titer, of a lysate.
After the titer is obtained, the phage will be amplified by using larger volumes and bigger
plates. The goal to find a phage has been met, but the phage must still be characterized.
1
http://www.encyclopedia.com/topic/bacteriophage.aspx, The Columbia Encyclopedia, Sixth Edition, 2008
http://en.wikipedia.org/wiki/Bacteriophage
3
http://en.wikipedia.org/wiki/Bacteriophage
4
http://www.zeuscat.com/andrew/personal/info/tphage/
5
http://www.phages.org/PhageInfo.html
6
http://www.phages.org/PhageInfo.html
2