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

MS2 Fd, M13T2

For lecture only BC Yang

Historical context A century ago, Hankin (1896) reported that the waters of t

he 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 pfu: plaque forming unit Title: define pfu in a phage suspension moi: multiplicity of infection, the ration of phag

e particles to bacteria eop: efficiency of plating, the ration of the plaq

ue titer to the number of phage particles Prophage: state of phage co-existing with host Lysogenic bacteria: term of bacteria carrying p

rophage Phage conversion: phenotype change in lysogen

ic bacteria

For lecture only BC Yang

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 growthdemonstrate 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

1

2

3

4

5 6 7

For lecture only BC Yang

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

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

For lecture only BC Yang

Adsorption and DNA injection

A random collision, protein/prote

in interaction

Affected by Ca++, Mg++, or trypto

nphanetc.

Receptor specific (outer membra

ne protein lamB for lambda; sex

pili for Q)

DNA is the major material entering bacterial

Lysozme like activity, core boring through the cell wall

For lecture only BC Yang

Developmental gene expressionassay by protein synthesis

Early, in 5 min

Middle, in 10 min

LateIn 25 min

For lecture only BC Yang

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

Assembly of phageCan it happen automatically?

For lecture only BC Yang

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 D

NA into bacterial chromosome via short sites of homology (site-specific recombination) ---- prophage

For lecture only BC Yang

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 th

at cuts prophage DNA from bacterial chromosome Lytic cycle can start.

For lecture only BC Yang

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