Transcript Bacteriophage

Bacteriophage
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Prokaryotes as host
Subcellular structure without metabolic machinery
Double stranded DNA, single stranded DNA, RNA
Virulent phage vs. template phage
MS2
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T2
Fd, M13
BC Yang
Historical context
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A century ago, Hankin (1896) reported that the waters of
the Ganges and Jumna rivers in India had marked
antibacterial action (against Vibrio cholerae, restrict
epidemic) which could pass through a very fine porcelain
filter; this activity was destroyed by boiling.
Edward Twort (1915) and Felix d'Herelle (1917)
independently reported isolating filterable entities capable
of destroying bacterial cultures and of producing small
cleared areas on bacterial lawns.
It was F d'Herelle, a Canadian working at the Pasteur
Institute in Paris, who gave them the name
"bacteriophages"-- using the suffix phage (1922).
For lecture only
BC Yang
Glossary
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pfu: plaque forming unit
Title: define pfu in a phage suspension
moi: multiplicity of infection, the ration of
phage particles to bacteria
eop: efficiency of plating, the ration of the
plaque titer to the number of phage particles
Prophage: state of phage co-existing with host
Lysogenic bacteria: term of bacteria carrying
prophage
Phage conversion: phenotype change in
lysogenic bacteria
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BC Yang
plaque
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Plaques are clear zones formed
in a lawn of cells due to lysis by
phage.
At a low multiplicity of infection
(MOI) a cell is infected with a
single phage and lysed,
releasing progeny phage which
can diffuse to neighboring cells
and infect them, lysing these
cells then infecting the
neighboring cells and lysing
them, etc,
It ultimately results in a circular
area of cell lysis in a turbid lawn
of cells.
Dynamic process
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gal+
gal-
BC Yang
One step growth
demonstrate an eclipse period during which the DNA began
replicating and there were no free phage in the cell, a period
of accumulation of intracellular phage, and a lysis process
which released the phage to go in search of new hosts.
Ellis, E. L. and M. Delbrück (1939). The Growth of
Bacteriophage. J. Gen. Physiol. 22:365-384.
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BC Yang
Lytic cycle of phage
5
3
1
6
7
4
2
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BC Yang
Kinetics of phage infection
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0 min. Attachment of T2 to a susceptible E. coli cell
1 min. Inject DNA into cell
1-7 min. Transcribe and translate early genes
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block bacterial DNA synthesis and degrade host chromosomal DNA
block transcription of host mRNAs
block translation of host proteins
small amounts of early proteins produced (catalytic functions)
transcription from single phage genome
7-15 min. Replication of phage DNA
10-20 min. Translation of phage late proteins (structural)
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transcribed from new phage DNA (many copies of template)
need large amounts of these proteins to build new virions
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18-25 min. Assembly of new phage particles (end of eclipse
period)
 25 min. Lysis of host cell and release of progeny (end of
latent period)
For lecture only
BC Yang
Infection processes
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1.
2.
Attachment of virion to cell
3.
Early viral proteins synthesized (required for
genome replication)
4.
5.
6.
7.
Genome replication
Entry of viral nucleic acid into host cell (with
or without other virion components)
Late proteins synthesized (capsid proteins)
Assembly of progeny virions
Release of infectious progeny virions
BC Yang
Adsorption and DNA injection
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A random collision,
protein/protein interaction
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Affected by Ca++, Mg++, or
tryptonphanetc.
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Receptor specific (outer
membrane protein lamB for
lambda; sex pili for Qb)
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DNA is the major material
entering bacterial
 Lysozme like activity, core
boring through the cell
wall
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BC Yang
Developmental gene expression
assay by protein synthesis
Early, in
5 min
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Middle,
in 10 min
Late
In 25 min
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Host gene shut-off
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Altering RNA
polymerase activity
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Change the translation
apparatus (translation
of the MS2 phage RNA
with ribosome of T4infected cells reduced
by 88%)
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Degradation of host
DNA
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XP10
BC Yang
Assembly of phage
Can it happen automatically?
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BC Yang
Lysogenic cycle
Lysogenic Cycle: Lambda as an example
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lambda integrase and lambda repressor cI
synthesized due to activation of the
transcription of their genes by cII.
 cI repressor turns off phage transcription
 integrase catalyzes integration of lambda
DNA into bacterial chromosome via short
sites of homology (site-specific
recombination) ---- prophage
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BC Yang
Return to be a killer
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Prophage:
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Prophage can be excised when host response
system to potentially lethal situations:
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Bacterium is now immune to infection by another phage,
because repressor continuously produced ----- new phage DNA
can be injected into cell and is circularized but is not
transcribed or replicated.
if host DNA damaged
one reaction by host cell is to activate a protease
protease also cleaves repressor
Phage DNA now transcibed including a gene for an enzyme
that cuts prophage DNA from bacterial chromosome
Lytic cycle can start.
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BC Yang
Application of phages
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Model system of
molecular biology
Cloning and
expression
Phage display system
Phage typing
Phage therapy:
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phage as natural, selfreplicating, selflimiting antibiotics.
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BC Yang