Bacteriophages
T-phage
Lemda phage
•
Suggested supplementary Reading
•
Discovery of phages
•
Morphology of Viruses
•
Classification of Viruses
•
Structure of TMV
•
Structure of T4 phages
The lifecycle of a bacteriophage
•
Lytic
cycle:
The process of a phage infecting a bacterium and
producing progeny is referred to as a lytic infection.
Some
phage, like T4, are only capable of lytic growth
.
•
Lysogenic cycle:
Some phage, like T4, are only capable of
lytic growth. Some phage are also capable of maintaining
their chromosome in a stable, silent state within the
The lifecycle of a bacteriophage
Temperate
phage:Phage that are capable of
both a lytic and lysogenic pathway are called
temperate phage.
e.g. P1 and lemdada are temperate phage.
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Figure 6.9
Virulent phages
only
undergo a lytic cycle
Temperate phages
can
follow both cycles
14-65
Prophage can
exist in a dormant
state for a long
time
It will undergo
the lytic cycle
This
process is
termed
8
PHAGE PLAQUES
~10
7HOST CELLS
~10 T4 AND
~10
7HOST CELLS
TOP AGAR
TOP
AGAR
AGAR
PLATE
CONFLUENT
GROWTH
INCUBATE
1.
ADSORPTION / ATTACHMENT
SPECIFICITY-RECEPTORS
2.
INJECTION / PENETRATION
SHEATH CONTRACTS
CHROMOSOME INJECTION
3.
SYNTHESIS OF COMPONENTS (REPLICATION)
TRANSCRIPTIONTRANSLATION ENERGY
PRECURSORS RIBOSOMES
PRODUCES: VIRAL mRNA
VIRAL DNA (RNA) CHROMOSOME
VIRAL STRUCTURAL PROTEINS
4.
ASSEMBLY / MATURATION
DNA PACKAGED
TAILS ADDED
INTACT VIRUSES PRODUCED
5.
LYSIS / RELEASE
T4 LYSOZYME
Life cycle Lytic phases
1. ADSORPTION / ATTACHMENT
SPECIFICITY-RECEPTORS: Phage identifies a
host bacterium by binding or absorbing to a
specific structure on the cell surface. J
Life cycle Lytic phases
2.
INJECTION / PENETRATION:
The tightly packed lambda DNA is
ejected from the phage and is taken up by the bacteria. When the l DNA
comes out of the phage head, the right end exits first. So “right” end of
the molecule exits the phage head first. In addition to LamB, l also uses
an inner membrane protein called PstM to gain entry to the cytoplasm.
How the l DNA physically traverses the peptidoglycan and periplasm and
gets through PtsM is not known.
Protecting lemda genome in bacterial cytoplasm- In capsid, lemda DNA is in linear
double stranded form. In the bacterial cytoplasm, dsDNA molecules are subject to degradation by exonucleases that need a free end to digest the DNA. The first event that happens to newly injected l DNA is that the DNA circularizes to prevent it from being degraded by host exonucleases. The DNA anneals at the cut cos sites that exist on both ends of the linear strand. Host encoded ligase seals the nick at either end generating a covalently closed circular lamda molecule.
3. SYNTHESIS OF COMPONENTS
Replication:
theta mode of
replication
-Upon injection into cytoplasm, the
initial mode of replication is theta mode of replication.
Rolling Circle Replication: Later in lytic development, lemda
switches to a second mode of replication called rolling circle
replication. The 5’ end of the cut plus strand is peeled away
from the intact strand.
The intact minus strand acts as a template for DNA
Polymerase which adds deoxyribonucleotides to the free 3’
OH of the cut plus strand.
Rolling circle replication produces long DNA molecules called
•
Rolling circle replication
of l DNA commences when an
endonuclease, en-coded by lemda exo, cuts one strand of
the covalently closed circular double-stranded DNA
molecule .
•
The cut strand is called the plus strand. The 5’ end of the
cut plus strand is peeled away from the intact minus
strand.
•
DNA polymerase adds deoxyri-bonucleotides to the free 3’
OH of the cut plus strand using the intact circular minus
strand as the template. This produces new plus strands
through a process of continu-ally elongating the original
plus strand.
•
The new plus strands are used as a template to synthesize
Assembly and Maturation
•
Capsid formation:
phage encoded proteins B,
C, and Nu 3 form a small initiator structure.
Host encoded GroEL and GroES proteins help
remodel proteins and protein complexes. The
major coat protein E is added to the structure
to form an immature phage head. Nu3
degrades, B is cleaved to B*, and C protein,
which is then cleaved into x1 and x2 protein to
form a mature phage head.
•
DNA Packaging :
lambda encoded terminase enzyme
binds to cos site and to the mature phage head.
Terminase cuts the cos site asymmetrically leaving a 12
bp single strand overhang, then inserts that end of the
DNA into the phage head.
•
As the ks along the concatomer of terminase enzyme
tracks along the concatomer of DNA, the DNA is
inserted into the head until terminase reaches a
second cos site.
•
The terminase cuts the DNA asymmetrically at the cos
site and packages the right end of the of the last bit of
DNA into the head.
•
The W and FII proteins are added to the base of the full
•
Tail construction
Tails are constructed from 12 gene products
beginning with J protein. The initiator complex
requires the J,I,L,K,H,G and M phage encoded
proteins. The major tail protein V is added. H
protein determines how long the tail will be.
Once the correct length is reached the U
•
Mature tails add spontaneously to the DNA
filled capsid.
Lysis/Release from cell
S protein forms a hole in the inner membrane.
This allows lambda encoded R protein, and
endolysin, to degrade the peptidoglycan cell
wall.
After the hole is formed, approximateley 100
intact lambda phage particles are released
into the environment.
Lysogenic Cycle
The establishment of an integrated lambda prophage occurs
in
four major steps
:
1. Circularization of Phage Chromosome
Linear phage DNA is injected into the host bacterial cell; the phage DNA is circularized by base pairing of its terminally redundant tails.
2. Site-specific recombination
The phage DNA is usually integrated or inserted at a specific site into the host chromosome as a prophage with the aid of integrase.
3. Repression of phage genome
The bacterium survives and multiplies; the prophage is replicated along with the host
chromosome.
4. Multiplication of prophase
14
REPRESSOR
PROPHAGE
BINARY FISSION
LYSOGENS;
LYSOGENIC;
PASSIVE REPLICATION
1.
Circularization of Phage Chromosome
Lysogenic cycle of lemda phage
Lambda DNA is a double stranded linear molecule having 5' cohesive ends, which can base pairn to produce a circular molecule. The circularization of DNA prevents its digestion by exonucleases.
2. Site-specific recombination
The phage DNA is usually integrated or inserted at a specific site into the host chromosome as a prophage with the aid of integrase, and host -encoded IHF (integration host factor ) protein . Lembda recombines into the phage chromosome using a specific site on the called Att P and a specific site on the bacterial chromosome called att B. When the lemda DNA is
