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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.

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Bacteriophage

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  1. 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

  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). For lecture only BC Yang

  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 For lecture only BC Yang

  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- For lecture only BC Yang

  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. 22:365-384.  For lecture only BC Yang

  6. Lytic cycle of phage 7 6 5 3 1 4 2 For lecture only BC Yang

  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) For lecture only BC Yang

  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 For lecture only BC Yang

  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 For lecture only BC Yang

  10. Developmental gene expressionassay by protein synthesis Early, in 5 min Middle, in 10 min Late In 25 min For lecture only BC Yang

  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 For lecture only BC Yang

  12. Assembly of phageCan it happen automatically? For lecture only BC Yang

  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 For lecture only BC Yang

  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. For lecture only BC Yang

  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. For lecture only BC Yang

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