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

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bacteriophage

Prokaryotes as host

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

Bacteriophage

Fd, M13

T2

MS2

For lecture only

BC Yang

historical context
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).

For lecture only

BC Yang

glossary
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

For lecture only

BC Yang

plaque
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-

For lecture only

BC Yang

one step growth
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. 

For lecture only

BC Yang

lytic cycle of phage
Lytic cycle of phage

7

6

5

3

1

4

2

For lecture only

BC Yang

kinetics of phage infection
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)

For lecture only

BC Yang

infection processes
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

For lecture only

BC Yang

adsorption and dna injection
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

For lecture only

BC Yang

developmental gene expression assay by protein synthesis
Developmental gene expressionassay by protein synthesis

Early, in 5 min

Middle, in 10 min

Late

In 25 min

For lecture only

BC Yang

host gene shut off
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

For lecture only

BC Yang

lysogenic cycle
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

For lecture only

BC Yang

return to be a killer
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.

For lecture only

BC Yang

application of phages
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.

For lecture only

BC Yang