Bactriophage Lambda

Part-I

Objectives
1. To know the temperate life cycle of bacteriophage lambda
2. How lambda phage chooses between the lytic and lysogenic life cycle
3. How the repressor protein helps to establish the lysogenic cycle

Summary

Lambda phage was discovered by Esther Lederberg in 1950. It has been used heavily as a model
organism, and has been a rich source for useful tools in molecular biology.

Lambda phage infects Escherichia coli K12 strain. The phage has a head, containing double-
stranded linear DNA flanked by 12-base-pair, single-stranded segments as its genetic material
(48,490 base pairs-40 genes), and a tail that can have tail fibers. The phage particle injects its
DNA into its host through the tail, and the phage will then usually enter the lytic pathway.
However, under certain conditions, the phage DNA may integrate itself into the host cell
chromosome in the lysogenic pathway. In this state, the λ DNA is called a prophage and stays
resident within the host's genome without apparent harm to the host, which can be termed a
lysogen when a prophage is present. The prophage is duplicated with every subsequent cell
division of the host. The phage genes expressed in this dormant state code for proteins that
repress expression of other phage genes. In response to UV ray or in presence of chemical
mutagens like hydrogen peroxide or mitomycin C, the activated prophage is excised from the
host cell DNA and enters its lytic pathway. The protein responsible for establishing the lysogenic
cycle of lambda is the product of gene CI and the protein that helps to follow the lytic cycle is
the product of cro gene.

Introduction
Bacteriophages are viruses that infect and replicate within bacteria. The term is derived from two
words 'bacteria' and ‘phage’ meaning ‘to eat’. Phages are widely distributed wherever bacterial
hosts are found such as soil or the intestines of animals. Sea water contains up to 9×10 8 virions/
milliliter and up to 70% of marine bacteria may be infected by phages. Phages have been used
for over 90 years as an alternative to antibiotics in Europe and called Phage therapy. Phage
infection is highly specific and there are about 10 phages for every type of bacteria. Viruses are
very specific about their hosts and depending on the host, they are classified as Animal virus,
Plant virus and Bacterial virus or bacteriophage.

Many viruses always follow lytic cycle whereas some follow either lytic or lysogenic and are
called temperate bacteriophages.

The lytic bacteriophage (T4) life cycle is composed of four phases:

 adsorption of the phage to the host and penetration of virus genetic material
 synthesis of virus nucleic acid and capsid proteins
 assembly of complete virions
 release of phage particles from the host

In the Lysogenic life cycle, the virus genome is integrated into the host genetic material after
infection. It is called a prophage. The temperate virus genetic material remains in the host cell
and reproduces in synchrony with the host for long periods in a relationship known as lysogeny.

In lysogeny, the prophage remains in the host but does not kill (lyse) the host cell; It may switch
to the lytic cycle at some later time and the switching to a lytic cycle is called induction. Most
bacteriophages are temperate indicating that this life strategy is advantageous

At adverse conditions, when number of host cells is low, 1 T4 phage infects one host releasing
300 new phages.
1 lambda phage infects one host→ Host produces 1000 daughter cells → Lambda emerges with
100 phages per cell = 100,000 new phages

Enterobacteria phage λ (lambda phage) is the most common and popular temperate
bacteriophage studied so far.

Family: Siphoviridae
Genus: λ -like viruses
Species: λ Phage

Figure: lambda phage under electron microscope

Infection: Bacteriophage λ binds to the target E. coli cell with the J protein in the tail tip to the
lamB gene product of E. coli. lamb gene is a part of the maltose operon and the protein forms a
porin molecule called maltoporin which helps in the entry of maltose in Gram –ve bacteria. The
linear phage genome is injected past the cell outer membrane through the maltoporin. Thus
infection of E.coli by lambda is facilitated in presence of maltose. The lambda genome is linear
DNA of ~49kbp flanked by 12-base-pair, single-stranded segments of DNA at both the ends.
These 12-base-pair G-C rich single-stranded segments are complementary to each other and thus
called cohesive ends or sticky ends. These ends help to circularize the lambda genome
immediately as it enters into the bacteria. The single-stranded nicks are ligated by host DNA
ligase. Host DNA gyrase puts negative supercoils in the circular chromosome, causing A-T rich
regions to unwind and drive transcription.

Figure: Lambda genome

Transcription of lambda genome

(i) Immediate early: Transcription starts from two strong constitutive promoters, PL and
PR producing the 'immediate early' transcripts. Initially the transcripts express the N
and cro genes, producing N, Cro proteins respectively. Transcription also starts at a
very slow rate from the PRM promoter producing cI transcript.

(ii) Delayed early: the delayed early genes flank the immediate early genes N and cro.
The gene product of N allows the RNA polymerase to read through the transcriptional
terminators tL and tR1into the two important delayed early genes, cII and cIII.
Antitermination- The N protein acts as an antiterminator and functions to extend the reading
frames that it is bound to beyond the termination sites. N protein binds to the two Nut (N
utilisation) sites in the mRNA, one in the N gene in the P L reading frame, and one in the cro gene
in the PR reading frame. N protein binds to nutR site as soon as it is transcribed and undergoes
conformational change. Then it binds to RNA polymerase. The binding of N protein to RNA pol
is facilitated by several host Nus proteins, A, B, E and G. This complex composed of RNA pol,
nutR sequence, N protein and Nus ABEG proteins skips through most termination codons. The
extended transcripts (the 'late early' transcripts) include the N and cro genes along with cII and
cIII genes, and xis, int, O, P and Q genes.

The outcome of infection, lytic or lysogeny, remains unresolved until the end of the delayed
early phase. By this time, the phage proteins required to take the decision are present in the
bacterial cell. The decision is influenced by the multiplicity of infection (MOI) and by the
nutritional status of the E. coli cell.

CII protein- The protein which plays an important role in the decision making is CII, the
activator of transcription of lambda genes that i) represses lytic function (by the CI protein) and
ii) catalyzes integration of the lambda genome into the host chromosome.

CII protein is synthesized as a 97 amino acid polypeptide. The polypeptide becomes active after
removal of the amino terminal methionine and the subterminal valine and in its tetrameric form.
CII is a DNA binding protein and with RNA polymerase it binds with a 20-25bp region of three
leftward promoters: PRE, PI and PAQ.

Cro protein binds to OR3 preventing access to the PRM promoter preventing expression of the cI
gene. On the other hand, cII gene product binds to another promoter and starts transcription of cI
gene. The cI gene product is called the repressor protein and needed to establish lysogeny. The
promoter is named PRE for promoter for repressor establishment. cII gene product also binds to
two other promoters PI and PAQ and initiates transcription. PI promoter expresses the integrase
enzyme which helps lambda genome to integrate into the bacterial chromosome. PAQ expresses an
antisense mRNA of Q mRNA and stops translation of Q protein which is required for the
expression of late genes. The cIII gene product protects the cII gene product from proteolysis by
FtsH (a membrane-bound essential E. coli protease) (hflA gene product, high frequency
lysogeny) protease by acting as a competitive inhibitor. This inhibition can induce a
bacteriostatic state, which favours lysogeny. Gp cIII also directly stabilizes the cII protein.

On initial infection, the stability of cII determines the lifecycle of the phage; stable cII will lead
to the lysogenic pathway, whereas if cII is degraded the phage will go into the lytic pathway.
Low temperature, starvation of the cells and high multiplicity of infection (MOI) are known to
favor lysogeny.

Now the big question comes… How l executes its two life cycles: LYTIC & LYSOGENIC?
The decision depends on the fate of the battle between two proteins, CI and Cro which compete
for the same binding sites (operators) on phage DNA

– If CI binds, represses synthesis of all genes = Lysogenic

– If Cro binds, represses synthesis of cI = Lytic

The operator region is subdivided into three similar but nonidentical regions of 17bps, O L1, OL2,
and OL3 for PL, and OR1, OR2 and OR3 for PR.

CI protein

CI protein or λ repressor is the product of cI gene. It is a 236 aa polypeptide which is inactive at

very low concentrations. This protein has two domains joined by a flexible linker region. Amino
terminal domain contains the DNA binding region (a helix turn helix domain) and the carboxy
terminal domains of two λ repressors join together to form a dimer at physiological
concentrations. A single dimer recognizes 17 basepair DNA sequence. λ repressor can both
activate and repress transcription. CI binds most favorably to O R1; binding here inhibits
transcription from PR. Binding of cI at OR1 stimulates an almost simultaneous cI binding to O R2
via cooperative binding or dimerization. This binding helps RNA polymerase to bind P RM and
hence stimulate its own transcription. When it is present at a much higher concentration, it also
binds to OR3, inhibiting transcription from PRM, thus regulating its own levels in a negative
feedback loop.

Figure: lambda repressor in dimeric form

Binding affinity of lambda repressor for the operator region OR1>OR2> OR3 and

Binding affinity of Cro for the operator region OR3> OR2>OR1

Cro protein

Cro is a 66 amino acid polypeptide and affinity of Cro for operator regions is completely
opposite of CI and this phenomenon helps lambda to select between lytic and lysogenic.
Thus Cro will inhibit cI expression but continue the transcription from P R and the phage will
follow Lytic Lifecycle. This is the lifecycle that the phage follows following most infections,
where the CII protein does not reach a high enough concentration due to degradation, so does not
activate PRE promoter. The 'late early' transcripts are synthesized including xis, int, Q genes for
replication of the lambda genome (OP). Cro dominates the repressor site, repressing synthesis of
cI from the PRM promoter.

Rightward transcription expresses the O, P and Q genes. O and P proteins are responsible for
initiating replication, and Q protein is another antiterminator which allows the expression of
head, tail and lysis genes from PR’. Transcription from the PR’ promoter can now extend to
produce mRNA for the lysis and the head and tail proteins. Structural proteins and phage
genomes self assemble into new phage particles.

Leftward transcription expresses the gam, red, xis and int genes. Gam and red proteins are
involved in recombination. Gam is also important in that it inhibits the host RecBCD nuclease
from degrading the 3’ ends in rolling circle replication. Int and xis are integration and excision
proteins which are vital to lysogeny.

Replication of lambda genome

The gene products of O and P, together with some of the host replication proteins and stress
proteins are required to replicate the phage DNA. The circular DNA replicates itself, initially in a
bidirectional manner similar to that used in
Escherichia coli (and called theta-replication) and then by rolling circle replication. It is not clear
why mode of replication is changed from theta to rolling circle mechanism. This results in the
production of multiple copies of the DNA joined together into concatemers. The heterotrimeric
protein exonuclease V, encoded by the bacterial RecB, RecC and RecD genes cleaves the 3’ ends
of the linear DNA generated by rolling circle model and thus inhibits conversion of theta mode
of replication to rolling circle mechanism until gam gene is transcribed. Gam protein binds to the
exonuclease and inhibits its action. Gam is not required if the host cell RecBCD is defective or
absent.

DNA packaging and assembly of bacteriophage particles

The head proteins assemble and produce a prehead. A host protein, gp groE, helps in further
maturation of the prehead. Two lambda proteins, Nu1 and A, together called terminase enzyme
help in the packaging of lambda genome into the prehead. Terminase, the packaging enzyme, is a
hetero-oligomer of polypeptides encoded by two leftmost genes Nu1 and A. Nu1 is a 181 aa
polypeptide and A is a 641 aa long polypeptide.

The encapsidation of the genome within the capsid during the assembly process of lambda phage
is ensured by three specific sequences called Cos sequences, CosN, CosB and CosQ. CosN
corresponds to the cleavage site of the terminase (carried out by the larger subunit of terminase
ie, gpA) on the DNA concatemer, which generates the cohesive ends of mature DNA and
initiates the encapsidation of the genome. CosB is the binding site of the terminase on the DNA
(gpNu1 is responsible for the binding). CosQ is however necessary as an arrest sequence of
encapsidation, it acts in line with the sites CosN and I2 (a sequence located between CosN and
CosB). The initiation of the cleavage at CosN is assisted by a host protein called the IHF,
Integration Host Factor which binds to a sub-sequence of CosB, I1. The DNA bends as the Nu1
protein binds to R1, 2 and 3 sites of CosB and A protein interacts with CosN. This interaction
triggers the endonuclease activity of the terminase subunit A protein. Following nicking
terminase remains bound to the left end of the lambda chromosome in a complex called complex
I. Then complex I binds to the portal protein (gpB) which helps in the entry of DNA into the
prohead. Terminase remains bound to the portal protein during the packaging until the
head is full, and introduces staggered nicks at the next cos site. During packaging, the head
enlarges and matures. The proteins D and FII attach to the outside of the filled capsid and
stabilize the head. The head with DNA about ~50kb then joins the preformed tail units and
complete the assembly.
The noncontractile tail is made of 32 rings of V protein. Each ring is composed of six
polypeptide subunits which form a hollow tube of 9nm diameter. The distal tip of the tail
contains at least 6 different polypeptides, of which J protein is responsible for adsorption to the
E. coli cell.

Lysis of the host cell

Lysis is brought about by three viral proteins: gps of R, S and Rz. S is an 8.5 kD inner membrane
protein. Homo-oligomers of S form holes in the inner membrane and
expose the peptidoglycan layer to R, which is an endolysin that degrades the peptidoglycan
layer of the periplasm. R is a soluble 17.5 kD transglycosylase which attacks the 1,6 glycosidic
bonds in the peptidoglycan cell wall. Rz is a 19kD membrane protein which is an endopeptidase
that cleaves oligopeptide crosslinks in the peptidoglycan cell wall. About 100 progeny phages
are liberated (the burst size is about 100).
Lysogenic cycle

Lysogenic Lifecycle: This is the lifecycle that the phage follows after a small number of
infections in specific conditions, where the cII protein reaches a high enough concentration due
to stabilisation and lack of degradation, and so activates its promoters.

The 'late early' transcripts continue being written, including xis, int, Q and genes for replication
of the lambda genome.

The stablized cII acts to promote transcription from the PRE, PI and PAQ (antiq) promoters.

The PAQ promoter produces antisense mRNA to the Q gene transcript, thereby switching off Q
production. In the absence of Q protein, transcription does not start from P R’ promoter and no
lytic or structural proteins are made.

The transcript produced from PRE promoter carries antisense mRNA to the cro gene of the PR
promoter transcript and a transcript of the cI gene. Hence the transcript turns down Cro
production whereas turns on CI repressor production. Production of cI protein leads to the
binding of CI to the OR1 and OR2 sites in the PR promoter, turning off cro and other early gene
expression. CI also binds to the PL promoter, turning off transcription there too. Lack of Cro
level the OR3 site unbound, so transcription from the P RM promoter may occur, maintaining levels
of cI proteins.

The PI promoter is located inside the xis gene which is followed by int gene. Thus the transcript
expresses the int gene, resulting in high concentrations of int protein. This protein integrates the
phage DNA into the host chromosome and much higher concentration of int than that of xis is
required to insert the λ genome into the host genome.

Transcription stops from the PL and PR promoters leading to no further production of CII and CIII
proteins. As CII and CIII concentrations decrease, transcription from the P AQ, PRE and PI also stop
since they are no longer needed. Only the PRM’ and PR’ promoters are left active, the former
producing cI protein and the latter a short inactive transcript. The genome remains inserted into
the host genome as the prophage in a dormant state.

Cro protein synthesis starts just after phage infection and thus before CI protein synthesis begins.
However, Cro protein binds OR less tightly than does lambda repressor. Thus it takes a higher
concentration of Cro protein in the cell to bind OR and block the binding of lambda repressor.

Integration of lambda genome into host chromosome

The integration of phage λ takes place at a special attachment (att) sites in the bacterial and
phage genomes. The sequence of the bacterial att site is called attB, between the gal and bio
operons, and consists of the parts B-O-B', whereas the complementary sequence in the circular
phage genome is called attP and consists of the parts P-O-P'. Both the attachment sites a
common core sequence of 15bp, GCTTT(TTTATAC)TAA- flanked by two dissimilar
sequences. The recombination always occurs within the bracketed 7bp sequence and the region
of homology is so short that recombination would not occur without the integrase protein. This
protein is a member of Y recombinase family since it has an active site tyrosine (Y) to which the
3’ phosphate end of the DNA is covalently attached. Integrase recognizs both attP and attB and
promotes recombination between them. It is called site specific recombination and requires both
the phage protein Integrase and the bacterial protein IHF (integration host factor). Both Int and
IHF protein bind to attP and form an intasome, a DNA-protein-complex designed for site-
specific recombination of the phage and host DNA. The original B-O-B’ sequence is changed by
the integration to B-O-P’-phage DNA-P-O-B’. The phage DNA is now part of the host’s
genome.

Maintenance of Lysogeny: Lysogeny is maintained solely by CI protein. CI represses

transcription from PL and PR while upregulating and controlling its own expression from PRM. It

CI binding to the PL operator is very similar, except that it has no direct effect on CI
transcription. As an additional repression of its own expression, however, CI dimers bound to
OR3 and OL3 bend the DNA between them to tetramerise. The presence of cI causes immunity to
superinfection by other lambda phages, as it will inhibit their PL and PR promoters.
Induction

Any situation where a lysogen undergoes DNA damage or the SOS response of the host is
otherwise stimulated leads to induction. The classic induction of a lysogen involved irradiating
the infected cells with UV light.

The host cell, containing a dormant phage genome experiences DNA damage due to a high stress
environment, and starts to undergo the SOS response. RecA (a cellular protein) detects DNA
damages and becomes activated. It is now called activated RecA, a highly specific co-protease.
Activated RecA binds to LexA (a transcription repressor) and activates its auto-protease activity,
which destroys LexA repressor. In the absence of LexA, transcription starts from all the DNA
repair protein encoding genes. In lysogenic cells this response is hijacked and activated RecA
stimulates CI autocleavage. This is because CI mimics the structure of LexA at the autocleavage
site.

Cleaved CI can no longer dimerise, and loses its affinity for DNA binding. The PR and PL
promoters are no longer repressed and switch on, and the cell returns to the lytic sequence of
expression events.

Excision of lambda genome from the host chromosome

xis and int regulates the insertion and excision of λ genome. xis and int are found on the same
piece of mRNA, so approximately equal concentrations of xis and int proteins are produced. This
results (initially) in the excision of any inserted genomes from the host genome.

Higher concentrations of xis than int result in no insertion or excision of phage genomes, the
evolutionarily favoured action - leaving any pre-inserted phage genomes inserted (so reducing
competition) and preventing the insertion of the phage genome into the genome of the host.

If enough Q protein accumulates in the cell (the production of which is suppressed by CI and
stimulated by N) then the phage will switch to the lytic cycle. Q protein binds a region of the
lambda DNA called qut, which is situated just downstream of the PR' promoter. Now transcripts
running from the PR promoter will continue past PR' onto the late
transcription phase structural genes (the head genes and tail genes) producing the proteins
needed to assemble phage particles.

Growth conditions regulate the stability of CII protein and thus the Lytic/lysogenic choice:

1. When the phage infects a population of bacterial cells that are healthy and growing
vigorously, it tends to propagate lytically, releasing progeny into an environment rich in
fresh host cells.
 2. When the conditions are poor for bacterial growth, however the phage is more likely to
form lysogen and sit tight as there will be very few host cells in the vicinity for any
progeny phage to infect.

Reason:

CII is very unstable protein in E.coli and is degraded by a specific protease called FtsH (HflB),
encoded by the hfl gene. Cells lacking hfl gene (and thus FtsH) almost always form lysogens
upon infection by lambda: in the absence of the protease, CII is stable and directs synthesis of
ample repressor. FtsH activity is itself regulated by the growth conditions of the bacterial cell.
i) If the growth condition is good, FtsH is very active, CII is destroyed efficiently,
repressor is not made, and phage tend to grow lytically
ii) If growth condition is poor, low FtsH activity, slow degradation of CII, repressor
accumulation and a tendency toward lysogenic development

Protein Function Overview

Cro; (Control of Repressor's Operator) Transcription inhibitor, binds OR3, OR2 and OR1 (affinity
OR3 > OR2 >= OR1, ie. preferentially binds OR3). At low concentrations blocks the P RM promoter
(preventing cI production). Cro inhibits its own production at a high concentration by blocking
the PR promoter. No cooperative binding is seen in case of cro like cI.

CI; (Clear 1) Transcription inhibitor, binds OR1, OR2 and OR3 (affinity OR1 > OR2 >= OR3, ie.
preferentially binds OR1). At low concentrations blocks the PR promoter (preventing cro
production) as well as PL promoter. Like Cro, cI also inhibits or downregulates its own
production through OR3 binding at high concentrations. Susceptible to cleavage by RecA* in
cells undergoing the SOS response.

CII; (Clear 2) Transcription activator and activates transcription from the PAQ, PRE and PI
promoters. CII is less stable due to susceptibility to cellular HflB (FtsH) proteases (especially in
healthy cells and cells undergoing the SOS response.

CIII;(Clear 3) HflB (FtsH) binding protein, protects cII from degradation by proteases.

N; (antiterminator) RNA binding protein and binds the mRNA (at Nut sites). Then it interacts
with RNApol and prevents its recognition of termination sites, so normal RNA polymerase
termination signals are ignored and RNA synthesis continues into distal phage genes.

Q; DNA binding protein and RNA pol cofactor, binds DNA (at Qut sites) and acts as an
antiterminator.

xis; (excisionase) excisionase enzyme, manages excision and insertion of phage genome into the
host's genome.
int; (integrase) Integrase protein, manages insertion of phage genome into the host's genome. In
conditions of low int concentration there is no effect. If xis is low in concentration and int high
then this leads to the insertion of the phage genome. If xis and int have high (and approximately
equal) concentrations this leads to the excision of phage genomes from the host's genome.

A, B, C, D, E, F, Z, U, V, G, T, H, M, L, K, I, J [A-F code for phage head genes, Z-J code for

phage tail genes]. The order shown here is as found on the genome, reading in a clockwise
direction]; these are the structural proteins, self assemble with the phage genome into daughter
phage particles.

S, R and RZ; lysis proteins and cause the host cell to undergo lysis at high enough concentrations.

O, P; DNA replication functions, promotes the specific replication of only the phage genome.

sib [not a protein, but a vital conserved DNA sequence]; Forms a stable hairpin loop structure in
transcribed mRNA beyond int and attracts degradation of mRNA by RNAaseIII.

attP [not a protein, but a conserved DNA sequence]; point of action of Int and Xis in integration
and excision of the phage genome into the host's genome. Corresponding attB found in the host's
genome at the point of insertion.

Lysogenic conversion

This occurs when a temperate phage induces a change in the phenotype of its host. Lysogenic
conversions often involve alterations in surface characteristics of its host.
E.g, infection of salmonella by epsilon phages change the activities of several enzymes involved
in construction of the carbohydrate component of the bacterium’s lipopolysaccharides, thereby
altering the antigenic properties of the bacterium. These changes also eliminate the receptor for €
phage making the bacterium resistant to infection by another € phage.
Corynebacterium diphtheriae produces the toxin of diphtheria only when it is infected by the
phage β ie the gene that codes for the toxin is carried by the phage, not the bacteria. Similarly,
Vibrio cholerae, which is normally a non-toxic strain, can become toxic when infected with the
phage CTXφ. phage CTXφ carries the cholera toxin gene.

Reference:

1. Molecular Cloning: A laboratory manual; Vol 1, (2.1-2.24) International Edition by

Joseph Sambrook and David W Russell , 2001
2. Molecular genetics of bacteria, 3rd Edition by Larry Snyder and Wendy Champness, ASM
Press, 2007.
3. Molecular Biology: Second edition; David Freifelder, Narosa Publishing House (2008)
4. Genetic studies of lysogenicity in E. coli by Esther M. Lederberg and Joshua Lederberg
GENETICS 38: 51 January 1953.
5. Microbiology- A Human Perspective 5th Edition by E W Nester, D G Anderson, C E
Roberts and M T Nester; Mc Graw Hill 2006.
Glossary:

Operator: An operator is a segment of DNA present upstream of a gene and acts as an

on/off switch for transcription ie where regulatory protein binds and modulates the
expression of the gene.
Promoter: a region of DNA upstream of a gene where RNA polymerase binds and
initiates transcription of that particular gene.
PRE : Promoter for repressor establishment.
PRM : Promoter for repressor maintenance.
SOS response: Activated RecA stimulates autocleavage of LexA, releasing repression of
DNA repair enzymes. This is called the SOS response.
RecA: RecA is a 38 kDa protein essential for the repair and maintenance of DNA
LexA: LexA is a repressor enzyme which represses SOS response genes under normal
conditions
Plaque: viral plaques are visible structure formed within a lawn of cells cultured in solid
nutrient medium. The viruses that follow lytic cycle, replicate and lyse the host cells,
generating round clear regions called plaques in the lawn of cells.
RNAse III- a nuclease that binds to and cleave double stranded RNA.
Cooperative binding- If a macromolecule has more than one ligand binding sites and
binding affinity towards ligands changes with the amount of ligand already bound, the
property is called cooperative binding. If the binding of ligand at one site increases the
affinity for ligand at another site, the macromolecule exhibits positive cooperativity. On
the contrary if the binding of ligand at one site lowers the affinity for ligand at another
site, the protein exhibits negative cooperativity.

Quiz:

1. The two most important regulatory genes of λ are:

a) cI and cro b) cI and cII c) cII and cIII d) cII and cro

2. The important regulatory promoters of λ are:

a) PR, PL and PRM b) PR’, PL and PRM C) PR, PL and PRE d) PR, PR’ and PRE

3. PR and PL are --------------and-------------- promoters

a) Inducible weak b) Constitutive, weak c) Inducible, strong d) Constitutive, strong

4) Lytic growth preceeds when:

a) PR and PL remain switched off, while PRM is kept switched on.
b) PR and PL remain switched on, while PRM is kept switched off.
c) PR , PL and PRM are switched on.
d) PR , PL and PRM are switched off.

5) Lysogenic growth is a consequence of:

a) PR and PL being switched off and PRM switched on.
b) PR and PL being switched on and PRM switched off.
c) All three switched on
d) All three switched off.

6) ----------- gene codes for λ repressor.

a) cI b) cII c) cIII d) Hfl

7) PRE is a weak promoter because it has a very poor -----------sequence.

a) -10 b) -35 c) UP element d) -10 and -35

8) ‘c’ of cI, cII, CIII stands for

a) Constitutive b) clear c) common d) consequitive

9. Which protein in lambda inhibits the change in mode of Replication from theta to
rolling circle?
a) LexA b) SSB c) RecBCD d) Gam

10. Which protein prevents the activity of RecBCD?

a) LexA b) SSB c) RecBCD d) Gam

FAQs

1. What is lysogeny? what are prophages? what are lysogens?

When the virus genome is integrated into the host genetic material after infection
and remains as a part of the bacterial genome and reproduces in synchrony with
the host for long periods, the phenomenon is called lysogeny. The viral genome is
called a prophage. The host cell is called a lysogen.

2. How does the lambda phage enter into its host?

The protein present at the tip of the tail of lambda phage (J protein) interacts with
maltose receptor present on the E. coli cell surface. Interaction changes the
conformation of tail proteins and the DNA genome enters into the cell from the
head.
3. What is the difference between Temperate phages and T phages?

T phages always follow lytic cycle ie they lyse the host cell after infection. The ‘t’
gene product is responsible for lysing the host cell.

Temperate phages either follow the lytic cycle or lysogenic cycle depending on
the MOI and nutrient environment.

4. What is cos site in lambda genome? What is its role?

Lambda genome is composed of about 49kbp long double stranded DNA flanked
by 12-base-pair, G-C rich single-stranded segments of DNA at both the ends.

5. What is lambda repressor? How cII gene product establishes lambda repressor in
cell?

The gene product of cI gene is called the lambda repressor. This protein represses
the lytic growth of the phage and establishes the lysogenic cycle. cII gene product
initiates transcription of cI gene from the promoter PRE.

6. Why the lambda repressor protein and the cII and cIII gene products are called 'C'
proteins?

Three proteins synthesized from cI, cII and cIII genes are required to establish
lysogeny. Hence, mutation in any of these three genes will lead to the phage towards
lytic growth and formation of plaque. Plaque is a clear zone in the bacterial lawn and
thus the genes are represented by ‘c’ for clear.

7. What is the role of cIII gene product in lambda phage?

The cII gene product is susceptible to proteolysis by bacterial protease. The cIII gene
product helps or protects the cII gene product from degradation.

8. Where does the attachment site in lambda genome present?

The attachment site is present after the int gene and before the RNase III cleavage
site.

9. How does the nutrient rich medium help the lambda phage to choose the lytic
cycle?

When the host cells are growing in nutrient rich medium, the protease called FtSH
degrades the cII gene product. In the absence of this protein transcription of cI gene
do not start.

10. At what site of E.coli genome does the lambda genome get incorporated?
 The lambda genome integrates between the gal and bio operon of E.coli genome.

Assignments

1. How does the operator region help in maintaining lysogeny?

2. How does the repressor regulate its own synthesis in the lysogenic state?

3. Why PRE is called promoter for repressor establishment?

4. What is cooperative binding? How does it help in maintaining lysogeny?

5. How lysogeny is established when Cro and CI proteins are present in same
amount?

Synopsis

Bacteriophage Lambda is a temperate phage which follows either lytic or lysogenic cycle. High
concentration of Cro protein leads the lambda genome towards the lytic cycle whereas high conc.
of CI protein favours the lysogenic cycle. New phages are generated and released from the host
cells durin the lytic cycle and lambda genome gets incorporated into the host cell in lysogeny.
One of the key players of lysogeny is CII protein which acts as an activator of lambda genes that
represses lytic function and catalyzes integration of the viral DNA into the host chromosome. CII
activates the synthesis of CI and integrase protein. Integrase helps in the integration of lambda
genome into the host cell and CI prevents the synthesis of Cro protein. Thus lysogeny is
established when host cells are growing in nutrient rich medium since accumulation of CII
occurs in healthy cells. High MOI also favours the lysogenic cycle.

Case study

Bacteriophage lambda was first isolated in the year 1951 by Esther Lederberg. She was working
with E.coli mutants (recipient cell) which survived treatment with intensive UV irradiation, grew
normally in pure culture but died when conjugated with another E.coli K12 strain (donor cell).
Separately UV exposure to the donor cell caused lysis of the cell. The donor bacteriophage was
found to be naturally lysogenic for bacteriophage lambda. During conjugation, the DNA of the
lysogenic strain was transferred to the recipient cell. The lambda genome entered into a
repressor-free cell and established a cycle of lytic growth, generating a batch of progeny particles
that could lytically infect the remaining nonlysogenic cells in the culture.