Bacteriophage

1
Bacteriophage

• Prokaryotes as host
• Subcellular structure without metabolic machinery
• Double stranded DNA, single stranded DNA, RNA
• Virulent phage vs. template phage

Fd, M13
MS2
T2
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2
Historical context

• 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).

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3
Glossary

• 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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4
plaque

• 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

gal
gal-
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5
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. 22365-384. 
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BC Yang
6
Lytic cycle of phage
7
6
5
3
1
4
2
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7
Kinetics of phage infection

• 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
• 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)
• 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)

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8
Infection processes

• Attachment of virion to cell
• Entry of viral nucleic acid into host cell (with
  or without other virion components)
• Early viral proteins synthesized (required for
  genome replication)
• Genome replication
• Late proteins synthesized (capsid proteins)
• Assembly of progeny virions
• Release of infectious progeny virions

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9
Adsorption and DNA injection

• A random collision, protein/protein interaction
• Affected by Ca, Mg, or tryptonphanetc.
• Receptor specific (outer membrane protein lamB
  for lambda sex pili for Qb)

• DNA is the major material entering bacterial
• Lysozme like activity, core boring through the
  cell wall

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10
Developmental gene expressionassay by protein
synthesis
Early, in 5 min
Middle, in 10 min
Late In 25 min
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11
Host gene shut-off

• Altering RNA polymerase activity
• Change the translation apparatus (translation of
  the MS2 phage RNA with ribosome of T4-infected
  cells reduced by 88)
• Degradation of host DNA

XP10
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12
Assembly of phageCan it happen automatically?
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13
Lysogenic cycle

• 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

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14
Return to be a killer

• 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
• 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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15
Application of phages

• Model system of molecular biology
• Cloning and expression
• Phage display system
• Phage typing
• Phage therapy
• phage as natural, self-replicating, self-limiting
  antibiotics.

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BC Yang