Lambda phage

Enterobacteria phage λ (lambda phage, coliphage λ, officially Escherichia virus Lambda) is a bacterial virus, or bacteriophage, that infects the bacterial species "Escherichia coli" ("E. coli"). It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.
The phage particle consists of a head (also known as a capsid), a tail, and tail fibers (see image of virus below). The head contains the phage's double-strand linear DNA genome. During infection, the phage particle recognizes and binds to its host, "E. coli", causing DNA in the head of the phage to be ejected through the tail into the cytoplasm of the bacterial cell. Usually, a "lytic cycle" ensues, where the lambda DNA is replicated and new phage particles are produced within the cell. This is followed by cell lysis, releasing the cell contents, including virions that have been assembled, into the environment. 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. The host is termed a lysogen when a prophage is present. This prophage may enter the lytic cycle when the lysogen enters a stressed condition.
Anatomy.
The virus particle consists of a head and a tail that can have tail fibers. The whole particle consists of 12–14 different proteins with more than 1000 protein molecules total and one DNA molecule located in the phage head. However, it is still not entirely clear whether the L and M proteins are part of the virion. All characterized lambdoid phages possess an N protein-mediated transcription antitermination mechanism, with the exception of phage HK022 
The genome contains 48,490 base pairs of double-stranded, linear DNA, with 12-base single-strand segments at both 5' ends. These two single-stranded segments are the "sticky ends" of what is called the "cos" site. The "cos" site circularizes the DNA in the host cytoplasm. In its circular form, the phage genome, therefore, is 48,502 base pairs in length. The lambda genome can be inserted into the " E. coli" chromosome and is then called a prophage. See section below for details.
Life cycle.
Infection.
Lambda phage is a non-contractile tailed phage, meaning during an infection event it cannot 'force' its DNA through a bacterial cell membrane. It must instead use an existing pathway to invade the host cell, having evolved the tip of its tail to interact with a specific pore to allow entry of its DNA to the hosts.
On initial infection, the stability of cII determines the lifestyle 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 (see later discussion).
N antitermination.
This occurs without the N protein interacting with the DNA; the protein instead binds to the freshly transcribed mRNA. Nut sites contain 3 conserved "boxes", of which only BoxB is essential.
Lytic life cycle.
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 its promoters.
Rightward transcription.
Rightward transcription expresses the "O", "P" and "Q" genes. O and P are responsible for initiating replication, and Q is another antiterminator that allows the expression of head, tail, and lysis genes from "PR’".
Q antitermination.
Q is similar to N in its effect: Q binds to RNA polymerase in "Qut" sites and the resulting complex can ignore terminators, however the mechanism is very different; the Q protein first associates with a DNA sequence rather than an mRNA sequence.
Leftward transcription.
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 vital to lysogeny.
Lysogenic (or lysenogenic) life cycle.
The lysogenic lifecycle begins once the cI protein reaches a high enough concentration to activate its promoters, after a small number of infections.
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 (such as the structural and lysis genes) in order to prevent entry into the lytic cycle. These repressive proteins are broken down when the host cell is under stress, resulting in the expression of the repressed phage genes. Stress can be from starvation, poisons (like antibiotics), or other factors that can damage or destroy the host. In response to stress, the activated prophage is excised from the DNA of the host cell by one of the newly expressed gene products and enters its lytic pathway.
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, 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'. The integration itself is a sequential exchange (see genetic recombination) via a Holliday junction and 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. 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.
Induction.
The classic induction of a lysogen involved irradiating the infected cells with UV light. Any situation where a lysogen undergoes DNA damage or the SOS response of the host is otherwise stimulated leads to induction.
Multiplicity reactivation and prophage reactivation.
Multiplicity reactivation (MR) is the process by which multiple viral genomes, each containing inactivating genome damage, interact within an infected cell to form a viable viral genome. MR was originally discovered with phage T4, but was subsequently found in phage λ (as well as in numerous other bacterial and mammalian viruses). MR of phage λ inactivated by UV light depends on the recombination function of either the host or of the infecting phage. Absence of both recombination systems leads to a loss of MR.
Survival of UV-irradiated phage λ is increased when the E. coli host is lysogenic for an homologous prophage, a phenomenon termed prophage reactivation. Prophage reactivation in phage λ appears to occur by a recombinational repair process similar to that of MR.
Repressor.
The repressor found in the phage lambda is a notable example of the level of control possible over gene expression by a very simple system. It forms a 'binary switch' with two genes under mutually exclusive expression, as discovered by Barbara J. Meyer.
The lambda repressor gene system consists of (from left to right on the chromosome):
The lambda repressor is a self assembling dimer also known as the cI protein. It binds DNA in the helix-turn-helix binding motif. It regulates the transcription of the cI protein and the Cro protein.
The life cycle of lambda phages is controlled by cI and Cro proteins. The lambda phage will remain in the lysogenic state if cI proteins predominate, but will be transformed into the lytic cycle if cro proteins predominate.
The cI dimer may bind to any of three operators, OR1, OR2, and OR3, in the order OR1 > OR2 > OR3.
Binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2, an effect called cooperativity. Thus, OR1 and OR2 are almost always simultaneously occupied by cI. However, this does not increase the affinity between cI and OR3, which will be occupied only when the cI concentration is high.
At high concentrations of cI, the dimers will also bind to operators OL1 and OL2 (which are over 2 kb downstream from the R operators). When cI dimers are bound to OL1, OL2, OR1, and OR2 a loop is induced in the DNA, allowing these dimers to bind together to form an octamer. This is a phenomenon called "long-range cooperativity". Upon formation of the octamer, cI dimers may cooperatively bind to OL3 and OR3, repressing transcription of cI. This "autonegative" regulation ensures a stable minimum concentration of the repressor molecule and, should SOS signals arise, allows for more efficient prophage induction.
Lytic vs. lysogenic.
An important distinction here is that between the two decisions; lysogeny and lysis on infection, and continuing lysogeny or lysis from a prophage. The latter is determined solely by the activation of RecA in the SOS response of the cell, as detailed in the section on induction. The former will also be affected by this; a cell undergoing an SOS response will always be lysed, as no cI protein will be allowed to build up. However, the initial lytic/lysogenic decision on infection is also dependent on the cII and cIII proteins.
In cells with sufficient nutrients, protease activity is high, which breaks down cII. This leads to the lytic lifestyle. In cells with limited nutrients, protease activity is low, making cII stable. This leads to the lysogenic lifestyle. cIII appears to stabilize cII, both directly and by acting as a competitive inhibitor to the relevant proteases. This means that a cell "in trouble", i.e. lacking in nutrients and in a more dormant state, is more likely to lysogenise. This would be selected for because the phage can now lie dormant in the bacterium until it falls on better times, and so the phage can create more copies of itself with the additional resources available and with the more likely proximity of further infectable cells.
A full biophysical model for lambda's lysis-lysogeny decision remains to be developed. Computer modeling and simulation suggest that random processes during infection drive the selection of lysis or lysogeny within individual cells. However, recent experiments suggest that physical differences among cells, that exist prior to infection, predetermine whether a cell will lyse or become a lysogen.
As a genetic tool.
Lambda phage has been used heavily as a model organism, and has been a rich source for useful tools in microbial genetics, and later in molecular genetics. Uses include its application as a vector for the cloning of recombinant DNA; the use of its site-specific recombinase (int) for the shuffling of cloned DNAs by the gateway method; and the application of its Red operon, including the proteins Red alpha (also called 'exo'), beta and gamma in the DNA engineering method called recombineering. The 48 kb DNA fragment of lambda phage is not essential for productive infection and can be replaced by foreign DNA. Lambda phage will enter bacteria more easily than plasmids making it a useful vector that can destroy or can become part of the host's DNA. Lambda phage can be manipulated and used as an anti-cancer vaccine, nanoparticle, targeting human aspartyl (asparaginyl) β-hydroxylase (ASPH, HAAH). Lambda phage has also been of major importance in the study of specialized transduction.