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Prokaryotes as host
Subcellular structure without metabolic machinery
Double stranded DNA, single stranded DNA, RNA
Virulent phage vs. template phage
For lecture only
MS2 T2
Fd, M13
BC Yang

 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

 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
For lecture only
BC Yang

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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
For lecture only gal
+ gal
-
BC Yang

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.
For lecture only
Ellis, E. L. and M. Delbrück (1939). The Growth of
Bacteriophage. J. Gen. Physiol. 22:365-384.
BC Yang

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For lecture only
BC Yang

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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
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 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)
 transcribed from new phage DNA (many copies of template)
 need large amounts of these proteins to build new virions
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

For lecture only
1.
Attachment of virion to cell
2.
Entry of viral nucleic acid into host cell (with or without other virion components)
3.
Early viral proteins synthesized (required for genome replication)
4.
Genome replication
5.
Late proteins synthesized (capsid proteins)
6.
Assembly of progeny virions
7.
Release of infectious progeny virions
BC Yang

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A random collision, protein/protein interaction
Affected by Ca ++ , Mg ++ , or tryptonphanetc.
Receptor specific (outer membrane protein lamB for lambda; sex pili for Q b
)
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DNA is the major material entering bacterial
Lysozme like activity, core boring through the cell wall
For lecture only
BC Yang

assay by protein synthesis
For lecture only
Early, in
5 min
Middle, in 10 min
Late
In 25 min
BC Yang

 Altering RNA polymerase activity
 Change the translation apparatus (translation of the MS2 phage RNA with ribosome of T4infected cells reduced by 88%)
 Degradation of host
DNA
XP10
For lecture only
BC Yang

For lecture only
Can it happen automatically?
BC Yang

Lysogenic Cycle: Lambda as an example
 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
For lecture only
BC Yang

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Prophage:
 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.
Prophage can be excised when host response system to potentially lethal situations:
 if host DNA damaged
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 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.
For lecture only
BC Yang

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Model system of molecular biology
Cloning and expression
Phage display system
Phage typing
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Phage therapy: phage as natural, selfreplicating, selflimiting antibiotics.
For lecture only
BC Yang