Bacteriophage replication and life cycle

1. Bacteriophages
Dr. Harinatha Reddy A.
Assistant Professor
Department of Life Sciences

2.  Bacteriophage is a virus that infects and replicates within a
bacterium.
 Edward Twort (1915) and Felix d'Herelle (1917) independently
isolated filterable entities capable of destroying bacterial cultures
and of producing small cleared plaques on bacterial colonies.
 The name Bacteriophage is given by Felix d'Herelle (1922).
 Bacteriophage is a complex bacterial viruses with both heads and
tails are said to have binal symmetry because they possess a
combination of icosahedral (the head) and helical (the tail)
symmetry.

3.  Majority of Bacteriophages contain ds DNA as a
genetic material.
 T1 to T7 and λ phage contain ds DNA.

4.  Enterobacteria phage T4 is a bacteriophage that infects E.
coli bacteria.
 The T4 phage is a member of the T-even phages, a group
including enterobacteriophages T2 and T6.
 T4 is capable of undergoing only a lytic lifecycle and not
the lysogenic lifecycle.
T4 Phase:

5. Virus particle structure:
 T4 is a relatively large phage, at approximately 90 nm wide and
200 nm long.
 The DNA genome is held in an icosahedral head, also known as a
capsid.
 Head contain 2000 capsomers.

6.  The phage consists of a long helical tail which is
connected to head with a collar.
 The T4’s tail is hollow tube so that it can pass its
nucleic acid into the cell it is infecting after attachment.
 Tail contain 144 protein subunits arranged in 24 rings.
The

7. Hexagonal base plate attached to end of tail.
The base plate contains six spikes or tail fibres at its six
corners.
 The tail attaches to a host cell with the help of tail fibres. The
tail fibres are also important in recognizing host cell surface
receptors.
 Tail fibre have lysozyme enzyme.

8. Lambda phage:
 Enterobacteria phage λ (coliphage λ) is
a bacteriophage, that infects the bacterial
species E. coli.
 λ phage contain linear or circular ds
DNA as genetic material 17 µm length
packed in a Icosahedral capsid.
 The capsid 55 nm in diameter consisting
of 300-600 capsomers.

9.  The head is joined to a non-contractile 180 µm long
tail by a connector.
 Tail sheath absent in lambda phage.
 Single tail fibre having lysozyme activity crating
hole on host.

10. DNA and Gene Organization of Phage Lambda:
 Lambda DNA is a linear and double stranded duplex of about 17 µm in
length.
 It consists of 48, 514 base pairs of known sequence.
 Both the ends of 5′ terminus consists of 12 bases which extend beyond
the 3′ terminus nucleotide.
 This results in single stranded complementary region commonly called
cohesive ends.
 The 12 nucleotides of cohesive ends are responsible for circularization
after injection of phage DNA into E. coli cell where the bacterial enzyme,
i.e., E. coli DNA ligase, converts the molecule to a covalently sealed
circle.

11. Life cycle of Bacteriophage Lambda:
 λ have either lytic or lysogenic cycle, depending on the
environment.
 In the lytic cycle, λ phage replicate rapidly and cause lysis
of the host cell.
 In the lysogenic cycle, the viral DNA circularizes and
integrates into the host DNA.

12. Infection:
 Bacteriophage λ binds to an E. coli cell by means of its J protein in the tail
tip.
 The J protein interacts with the maltose outer membrane porin (the
product of the lamB gene) of E. coli.
 The linear phage genome is injected through the outer membrane.
Circularization of Phage DNA:
 The DNA passes through the mannose permease complex in the inner
membrane encoded by the manXYZ genes and immediately circularises in
the cytoplasm by using unpaired12-base sticky (cohesive) ends.

 The single-strand viral DNA ends are ligated by host DNA ligase.

14. Lambda phage DNA injection into the cell
membrane using Mannose permease
complex a sugar transporting system.

15. The eclipse or Latent period:
 The DNA is released in the host cytoplasm, it is not
degraded by the nuclease enzymes of host cell.
 This is because of glycosylated hydroxymethyl cytosine
instead of cytosine in the DNA of phage.
 After enter in to cell the DNA of phage takes over the
charge of cell machinery and supress all cellular activities
such as synthesis of DNA, RNA, and proteins etc.

16.  On the basis of transcription the genes are grouped into
three classes:
 Immediate early genes: (N and cro).
 Delayed early genes: located left to N gene Example:
cIII, gam, red, xis and int.
 And right to cro example: cII, O, P and Q.
 Late genes: S, R, A , J genes.
• The Cro protein specified by bacteriophage lambda is a repressor of
the genes expressed early in phage development and is required for
a normal late stage of lytic growth.
• Lytic cascade: Cro GENE PRODUCT turns off CI gene, leads to late
gene expression

17. Genes are clustered by function in the
lambda genome
Recombination Control region Replication Lysis
Virus head
&tail
oR
Pint PL PRM PR PRE PR‘
att
int
xis
red
gam
cIII N cI cro cII O P Q S R A…J
promoter
operator
terminator
cos

18.  Transcription starts from the expression of N and cro genes, producing
N, Cro proteins.
 Cro binds to operator of PRM (PRM, RM stands for repressor maintenance)
promoter, preventing expression of the cI gene.
 After synthesis, gpN binds to nutL and nutR sites (N utilization sites)
present at left and right side of the promoters.
 When RNA polymerase moves along with the DNA, it picks up the gpN.
 The gpN acts as anti-terminator and controls the expression of most vital
function.

19.  Lambda repressor coded by the cI gene.
 The repressor protein a dumbbell shape with two binding site
one binding to DNA, while the other site binds with another
repressor molecule to generate a dimer.
 The choice between lysis and lysogeny is governed largely by
the interactions of five regulatory proteins called CI, CII, Cro,
N, and Q gene products.

20.  The CI, CII, and Cro proteins are regulatory proteins.
 The CI and Cro proteins are repressors, and the CII
protein is an activator.
 The Q proteins interact directly with the E. coli RNA
polymerase to permit transcription of phage DNA genome.
 This activity of the N and Q proteins is referred to as
antitermination.

21. oR
Pint oL
PL PRM PR PRE PR‘
tR3
tL1 tR1 tR2 t6S
att
int
xis
red
gam
cIII N cI cro cII O P Q S R A…J
Cro Cro Q
Lytic functions
Replication proteins
Viral head & tail proteins
Lytic pathway:
Lytic cascade: Cro turns off cI gene, Q protein action leads to late gene
expression

22.  Once sufficient N protein is synthesized, it interacts with
RNA polymerase and induce transcription of Q gene and
genes for proteins needed in viral replication.

23. lysogenic pathway:
 The lysogenic pathway is governed by another immediate-early
gene product, the CII protein.
 The CII protein is an activator that stimulates transcription from
two additional promoters, PRE and Pint.
 The transcript from PRE (Transcription for repressor) includes the
cI gene that encodes the CI protein, which is a repressor.
 If synthesized early enough CI protein, this repressor is capable of
suppressing virtually all bacteriophage transcription of genes.
 The transcript from Pint (promoter for the integration) includes
genes required for the integration of viral DNA into the host
chromosome through site specific recombination.

24. +
oR
Pint oL
PL PRM PR PRE PR‘
tR3
tL1 tR1 tR2 t6S
att
int
xis
red
gam
cIII N cI cro cII O P Q S R A…J
CIII CII
CI
+
Repressor
PRE = promoter for
repression
establishment
Int
tint
CII
Lysogeny: CII and CIII stimulate expression of CI to make repressor

25.  The lysogeny/lysis decision by the nutritional status of the host
cell.
 The CII protein is subject to rapid degradation by E. coli proteases.
 The proteases are more abundant when the cell is growing rapidly in
a rich medium, so that under these conditions the absence of CII
(activator) limits CI (repressor) production and the scale tips in
favor of lysis.
 When E. coli cells are starved, CII protein is elevated and the
resulting production of CI protein favours the lysogenic path.
 In the lysogenized state the phage is referred to as a prophage, the
lysogenic state can continue for countless cell generations.
 The prophage to emerge from the lysogenic state is a sudden
reduction in the CI protein concentration

26. Prophage integration:
 The integration of phage λ takes place at a special attachment site in
the bacterial and phage genomes, called attλ.
 The sequence of the bacterial att site is called attB, between the gal
and bio operons, whereas the complementary sequence in the
circular phage genome is called attP .
 The integration requires both the phage protein Int and the bacterial
protein IHF (integration host factor).
 Both Int and IHF bind to attP and form an intasome, a DNA-
protein-complex designed for site-specific recombination of the
phage and host DNA.

28.  In latent period early proteins are synthesized, some early
proteins are used as enzymes for viral DNA replication.
 The newly synthesized viral DNA molecules direct the
formation of new type of proteins called late proteins.
 Majority of late proteins are viral coat proteins.

29. Maturation:
 Head and tail formation start separately the protein
components aggregate around the DNA and form the
head of the phage.
Lysis or release:
 After formation of new bacteriophages the host
bacterial cell bursts and the phage particle are
released.