Bacteriophage T4 and Bacteriophage lambda

1. Bacteriophages
T4 and Lambda phages
Dr. Harinatha Reddy M.sc, Ph.D.
biohari14@gmail.com
Department of Microbiology
Sri Krishnadevaraya University
Anantapur, A.p. India

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 (Largest phase) is a member of the T-even
phages.
 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.
144 protein subunits arranged in 24 rings.

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.

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 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 creating
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 unpaired bases which extend
beyond the 3′ terminus nucleotide.
 This results in single stranded complementary region commonly called
cohesive ends.
 3 5
 5 3
 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 E.
coli cell.
 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.

13. 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.

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

16. 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.

17.  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

18. Genes are clustered by function in the
lambda genome
Recombination Control region Replication Lysis Virus
head&tail
Pint PL PRM PR PRE
att
int
xis
red
gam
cIII N cI cro cII O P Q S R A…J
promoter
operator
terminator
PRM, (promotor for repressor maintenance)
PRE (Promotor for repressor)
Pint (promoter for the integration)

19.  Transcription starts from the expression of N and cro genes, producing
N, Cro proteins.
 Cro binds to operator of PRM 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 genes.

20.  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, CIII,
Cro, N, and Q gene products.

21.  The CI and Cro proteins are repressors, and the CII and
CIII 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.

22. oR
Pint PL PRM PR PRE ‘
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 and N protein action leads to late
gene expression

23.  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.

24. lysogenic pathway:
 The lysogenic pathway is governed by another Delayed-early gene
product, the CII protein.
 The CII protein along with the CIII is an activator that stimulates
transcription from two additional promoters, PRE and Pint.
 The transcript from PRE 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.

25. +
Pint PL PRM PR PRE ‘
att
int
xis
red
gam
cIII N cI cro cII O P Q S R A…J
CIII CII
CI
+
Repressor
Int
CII
Lysogeny: CII and CIII stimulate expression of CI to make repressor

26.  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.

27.  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

28. Prophage integration:
 The integration of phage λ takes place at a special attachment site in
the bacterial and phage genomes.
 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.

30.  DNA Replication in Lytic cycle:
Teta replication or Rolling circular replication.

31. Maturation:
 In lytic cycle 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.

32. THANK UUUUU…….