Lambda Phage

Lambda phage 
is a temperate bacteriophage that infects Escherichia coli.. It have alternative replication pathways: Lysogenic or Lytic pathways.

History of Lambda Phage

In 1950, Esther Lederberg, an American microbiologist, was performing experiments on E. coli mixtures. She happened to observe streaks of mixtures of two types of E. coli strains that seemed to have been nibbled on and had viral plaque. One E. coli strain had been treated with ultraviolet light, so the damage was more apparent. It was later determined that this was caused by bacterial viruses, which replicated and spread resulting in cell destruction. The discovery led to the employment of Lambda phage as a model organism in microbial genetics as well as in molecular genetics.

Structure and Genome.

Lambda phage is a virus particle consisting of a Icosahedral head, measuring around 50-60 nanometers in diameter, containing double-stranded linear DNA as its genetic material, and a flexible tail that is around 150 nanometers long and may and may contain tail fibers.Lambda phage.jpg

Lambda contains a linear double stranded DNA genome of 48.5 KB.

Lambda phage exists in two form. Inside the phage it is linear double stranded DNA; inside host double stranded circular DNA molecule.


The life cycle of lambda

1. Lambda adsorption

Phage identify a host bacterium by binding or adsorption to a specific structure on the surface of the cell. Many different cell surface structures can be used as binding sites.

Lambda binds to an outer membrane protein called LamB via a protein that resides at the tip of the lambda tail called the J protein. LamB normally functions in the binding and uptake of the sugars maltose and Maltodextrin.


2. Lambda DNA Injection

Initially, lambda binds to LamB and the binding is reversible. This step requires only the lambda tail and the LamB protein.

Next, the bound phage undergoes a change and the binding to lag LamB becomes irreversible. This nature of the change is unknown but it requires that a phage head be attached to the phage tail.

Next, the lambda DNA is ejected from the phage and taken up by the bacterium. The DNA in the phage head is very tightly packed. If the condensed state of the phage DNA is stabilized, ejection if the DNA does not occur.

In addition to LamB, lambda also uses an inner membrane protein called Pstm to gain entry to the cytoplasm.


3. Protecting the lambda genome in the cytoplasm of the bacteria.

Lambda contains a linear double-stranded DNA molecule in its capsid. In the bacterium cytoplasm, dsDNA molecules are subjected to degradation by exonucleases that need a free end to digest the DNA.

The first event that happens to newly injected lambda DNA is that the DNA circularizes to prevent it from being degraded.

How circulation of DNA occurs?
Lambda has a specific site on its DNA, termed Cos sites (cohesive sites), which is used to circularize the DNA. The cos site is a 22bp sequence that is cut asymmetrically when the lambda DNA is packed. These cos sites are complementary to each other. A host enzyme, DNA ligase, seals the nicks at either end of the cos site generating a covalently closed, circular lambda genome. Basically two cos sites: cosL and cosR.

Restriction Alleviation.

This is a uniqueproperty of this virus.Lambda phage is very small.It mimics the methylation system and its own DNA is methylated. Because of this restriction enzyme will not cleave that DNA and are protected.

Restriction enzyme actually recognises a specific DNA sequence and cleaves the DNA on the both strands. The cut or digested DNA is sensitive to nucleases that degrade DNA. The modification part of the system is a protein that specifically modifies the DNA sequence recognise by the restriction enzyme and prevent the DNA from being digested.

This restriction or modification system allows a bacterium to tell DNA from its own species from foreign DNA.


4. What happens to the lambda genome after it is stabilized.

In case of lambda all the genes involved in specific functions are clamped together. SubattPxisint is the gene resp possible for recombination. The genome contain six major promoters. Function of Promoter is to promote transcription by binding to RNA polymerase.

PL – promoter leftward
PR – Promoter rightward
PRE – Promoter for repressor establishment.
PRM – Promoter for repressor maintenance.
PI – Promoter for integration
PR1 – Secondary rightward Promoter

CI – Repressor
Cro – Anti repressor
Q, N – Anti terminator proteins
Nut R – Nutrilisation site (rightward)
Nut L – Nutrilisation site (leftward)
tRI – Terminator right ward
tL – Terminator left ward
PL/OL – Promoter and operator (rightward)
PR/OR – Promoter and operator (leftward)
O, P – DNA synthesis
Paq – Promoter anti q
q nut – Q nutrilisation site

S helps in transcription of R to peptidoglycon, altimately weakening cell membrane for lysis.

After the genome is circularised and supercoiled (The host encoded enzyme, DNA gyrase, supwrcoils the lambda molecule), transcription begins from PL and PR. A series of genes known as early genes are transcribed and translated. These early genes are namely N and Cro. These genes products are the initial proteins needed for further phage development.

E coli RNA polymerase interacts with PL to give rise to short mRNA transcript that is translated into the N protein. Transcription from PR leads to Cro protein.

The N protein is able to extend transcription when RNA polymerase encounters a sequence in the DNA that tells it to stop. For that reason, N is called an anti termination protein. N allows RNA polymerase to transcript through tL and tR1 termination signals resulting in the synthesis of longer mRNA transcripts.

The longer transcript from PR encode the O, P and Cll proteins and a small amount of another anti terminator, the Q protein.

From PL, Clll the recombination proteins and a small amount of Xis and Int are made. Cll and Clll are reffered delayed early proteins. Cll helps in expression of promoters. Clll protects Cll from host proteases.

When N is bind to nut R1 / nut L1 the termination of R1 and L1 occurs. N protein anti terminates by binding to RNA polymerase after a specific base pair sequence, has been transcribed into mRNA. This sequence is called nut for Neutralization.

At this point, all of the players needed to make the lytic-lysogenic decision have been made. Cll and Clll are needed for lysogenic growth. The O and P proteins are used for replicating the lambda DNA.

Operater OR is further divided into OR3, OR2 and OR1. There are will be competition between Cro and Cl in competing in respect to binding. Cro likes to bind at OR3, if it bind it prevent binding of Cl. When N is bind to nutR1 and nutL1 the termination of R1 and L1 is not occurred and it continues dissociation. If no dissociation O,P,Q,S and R is produced. Function of O and P is to synthesise DNA, so phage DNA is synthesized.

DNA synthesis occurs on lytic cycle. Q protein is going to bind Qnut site, termination will not occur so expression of S and R occurs. S and R are responsible for lysis of the cell.


5. Lambda and the lytic-lysogenic decision:

At the most basic level, the decision depends on the amount of two phage encoded proteins called Cl and Cro, and their binding to their promoter control regions.

When Cl is bound, the expression of the lytic gene is repressed and the phage follows the lysogenic pathway. For this reason Cl is also known as Cl repressor or lambda repressor. The expression and binding of Cro will lead to lytic development. Cro is made from PR and Cl made from either PRE or PRM. Both Cro and Cl binds to the same DNA sequences called promoters.

Lambda contains two operators that bind Cro and Cl.
OR – overlaps PRM and PR promoters.
OL – overlaps with PL.

OR is the major player in the lytic-lysogenic decision, while OL is not a part of the decision. OR is composed of 17 base pair sequences called OR1, OR2 and OR3. Cl repressor binds to OR1, ten times better than it binds to OR2 and OR3. At increasing concentrations of Cl, it will bind to OR2 and eventually OR3.

When Cl repressor binds to OR, it stimulates the PRM Promoter and the production of Cl repressor and inhibits the PR promoter and the production of Cro, leading to lysogeny.

Cro binds to OR3 first, then OR2 and finally at high concentration to OR1. When Cro is bound to OR, it inhibits the PRM Promoter and the production of Cl, leading to lytic growth.

How does the phage switch between thses development pathways?

The major protein involved in the switch ia another phage -encoded protein called Cll. Cll activates the PRE and PI promoters. This leads to production of repressor, Cl and the integrase protein, which is also needed for lysogeny. Cll is unstable and rapidly degraded by the host-encoded HfIA protease. Inactivate Cll leads to lytic growth. Cll can be protected by the phage- encoded protein Clll. Active Cll leads to lysogrni. Cycle or growth.


The lambda lysogenic pathway

If active Cll prevails, it activates PRE and PI promoters leading to production of repressor CI and the integrase protein. Eventually Cl activates PRM ensuring that a continuous supply of CI is made.

Lambda recombines into the chromosomes using a specific site on the phage called attP and a specific site on bacterial chromosome called attB. When the lambda DNA is in the chromosome, it is bound by attL and attR, which are hybrid attP/attB sites.

Once the lambda DNA is recombined into the chromosome, it is replicated and stably inherited by daughter cells as part of the bacterial chromosome. It stays in quiescent.

What prevent the expression of the late genes coding for lytic function?

The expression of late genes is prevented by the action of the lambda repressor, Cl. Lambda repressor binding to the operator sequences OR and OL blocks transcription from PL and PR. Since PR is blocked, the lambda Q protein is not made and transcription of the late genes does not occur.


The lambda lytic cycle.

If enough of the Q proteins accumulates in the cell, RNA Polymerase will continue its transcription from the third promoter, PR1 located infront of the Q gene. This extends transcription in to the late genes located downstream of Q. The late genes encode the proteins needed to complete the lytic infection including the head, tail, and lysis proteins.

The Q proteins which is made from PR, when N is present is a second anti-termination protein. It acts on the qut site and allows transcription through tR1. Q is necessary for synthesis of the head and tail genes.

After the infecting lambda DNA has been converted to a double-stranded circular molecule, it replicates from a specific origin using both the phage encoded O and P proteins and bacterial encoded proteins. This is called theta replication. Later in lytic development, lambda switches to a second mode of replication called rolling circle replication. Rolling circle replication produces long DNA molecules containing multiple phage genomes called concatomers. These concatomers are packed int to phage heads and mature phages are produced. 

The lambda R and S proteins are required for lambda to release progeny phages into the environment. The R protein is an endolysin that degrades the peptidoglycan cell wall and allow phage to escape from the cell. The S proteins forms a hole in the inner membrane to allow the endolysin to gain excess to cellwall. After the hole is made, approx 100 intact lambda phage particles are released. The entire lytic cycle lasts~ 35 minutes.



Induction of lambda by the SOS System.

When a lambda lysogen is treated with UV ~ 35 minutes later te cells lyse and release phages. UV damages the DNA and triggers a cellular response called SOS response to deal with this damage. The single stranded DNA from the damaged DNA activates RecA. Activated recA interacts with Cl, leading to cleavage of Cl and induction of the lambda lysogen. Activated RecA also interact with LexA and leads to LecA. Inactivation which leads to expression of number of genes, whose products repair the DNA damage in the cell. Cleavage of Cl, leads to expression of the phage lytic genes and phage production. The rational for this response is that lambda does not want to risk staying in the cell that has DNA damaged and may not survive. 



If a cell is a lambda lysogen, another lambda phage that infects is not able undergo lytic development and produce phage. The incoming phage can inject its DNA, however the DNA is immediately shutdown and no transcription or translation of the lambda initiates. The Lysogens are immune to infection by another lambda phage particle, which is called superinfection. Superinfection is blocked because the lysogen is continuosly producing Cl repressor.

The lysogen actually produces more repressor than it needs to shut down one phage. This extra repressor binds to the superinfecting phage DNA at OL and OR and prevents transcription from PL and PR,


Applications of Lambda Phage 

The lambda phage has different applications, most of which are related to DNA cloning. This is because lambda phage can be used as a vector for generating recombinant DNA, which are combined DNA sequences that result from using laboratory techniques like molecular cloning to assemble genetic material from several sources. The site-specific recombinase of lambda phage can be used for shuffling cloned DNAs via the gateway cloning system, a molecular biology technique that ensures the effective transfer of DNA fragments between plasmids.

The lambda phage’s ability to mediate genetic recombincation is due to its red operon, which is a functioning unit of genomic DNA that has a cluster of genes controlled by a promoter or a single regulatory signal. This red operon can be expressed to yield the proteins red alpha (or exo), beta, and gamma, which can be used in recombination-mediated genetic engineering, a method commonly employed in bacterial genetics, generation of target vectors, and DNA modification.

Undoubtedly, the lambda phage is a powerful genetic tool that can be used in many important studies.

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