Lambda phage, also known as λ phage, (coliphage λ, scientific name Lambdavirus 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 infections, 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.
Contents
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 48,502 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.
The tail of lambda phages is made of at least 6 proteins (H, J, U, V, Stf, Tfa) and requires 7 more for assembly (I, K, L, M, Z, G/T). This assembly process begins with protein J, which then recruits proteins I, L, K, and G/T to add protein H. Once G and G/T leave the complex, protein V can assemble onto the J/H scaffold. Then, protein U is added to the head-proximal end of the tail. Protein Z is able to connect the tail to the head. Protein H is cleaved due to the actions of proteins U and Z.
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.
Bacteriophage Lambda 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, a porin molecule, which is part of the maltose operon.
The linear phage genome is injected through the outer membrane.
The DNA passes through the mannose permease complex in the inner membrane (encoded by the manXYZ genes) and immediately circularises using the cos sites, 12-base G-C-rich cohesive "sticky ends". The single-strand viral DNA ends are ligated by host DNA ligase. It is not generally appreciated that the 12 bp lambda cohesive ends were the subject of the first direct nucleotide sequencing of a biological DNA.
Host DNA gyrase puts negative supercoils in the circular chromosome, causing A-T-rich regions to unwind and drive transcription.
Transcription starts from the constitutive PL, PR and PR' promoters producing the 'immediate early' transcripts. At first, these express the N and cro genes, producing N, Cro and a short inactive protein.
Cro binds to OR3, preventing access to the PRM promoter, preventing expression of the cI gene. N binds to the two Nut (N utilisation) sites, one in the N gene in the PL reading frame, and one in the cro gene in the PR reading frame.
The N protein is an antiterminator, and functions by engaging the transcribing RNA polymerase at specific sites of the nascently transcribed mRNA. When RNA polymerase transcribes these regions, it recruits N and forms a complex with several host Nus proteins. This complex skips through most termination sequences. 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 discussed later.
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.
The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome (OP). Cro dominates the repressor site (see "Repressor" section), repressing synthesis from the PRM promoter (which is a promoter of the lysogenic cycle).
The O and P proteins initiate replication of the phage chromosome (see "Lytic Replication").
Q, another antiterminator, binds to Qut sites.
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.
Products of the genes S,R, Rz and Rz1 cause cell lysis. S is a holin, a small membrane protein that, at a time determined by the sequence of the protein, suddenly makes holes in the membrane. R is an endolysin, an enzyme that escapes through the S holes and cleaves the cell wall. Rz and Rz1 are membrane proteins that form a complex that somehow destroys the outer membrane, after the endolysin has degraded the cell wall. For wild-type lambda, lysis occurs at about 50 minutes after the start of infection and releases around 100 virions.
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'.
Pr is the promoter for rightward transcription, and the cro gene is a regulator gene. The cro gene will encode for the Cro protein that will then repress Prm promoter. Once Pr transcription is underway the Q gene will then be transcribed at the far end of the operon for rightward transcription. The Q gene is a regulator gene found on this operon, which will control the expression of later genes for rightward transcription. Once the gene's regulatory proteins allow for expression, the Q protein will then act as an anti-terminator. This will then allow for the rest of the operon to be read through until it reaches the transcription terminator. Thus allowing expression of later genes in the operon, and leading to the expression of the lytic cycle.
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 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome.
The stabilized cII acts to promote transcription from the PRE, PI and Pantiq promoters.
The Pantiq promoter produces antisense mRNA to the Q gene message of the PR promoter transcript, thereby switching off Q production. The PRE promoter produces antisense mRNA to the cro section of the PR promoter transcript, turning down cro production, and has a transcript of the cI gene. This is expressed, turning on cI repressor production. The PI promoter expresses the int gene, resulting in high concentrations of Int protein. This int protein integrates the phage DNA into the host chromosome (see "Prophage Integration").
No Q results in no extension of the PR' promoter's reading frame, so no lytic or structural proteins are made. Elevated levels of int (much higher than that of xis) result in the insertion of the lambda genome into the hosts genome (see diagram). Production of cI 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 leaves the OR3 site unbound, so transcription from the PRM promoter may occur, maintaining levels of cI.
Lack of transcription from the PL and PR promoters leads to no further production of cII and cIII.
As cII and cIII concentrations decrease, transcription from the Pantiq, PRE and PI stop being promoted 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 in a dormant state.
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. As discovered by Barbara J. Meyer, it forms a 'binary switch' with two genes under mutually exclusive expression.
The lambda repressor gene system consists of (from left to right on the chromosome):
cI gene
OR3
OR2
OR1
cro gene
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 an excellent tool first in microbial genetics, and then later in molecular genetics. Some of its 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, which can then be replicated by the phage. Lambda phage will enter bacteria more easily than plasmids, making it a useful vector that can either destroy or become part of the host's DNA. Lambda phage can also be manipulated and used as an anti-cancer vaccine that targets human aspartyl (asparaginyl) β-hydroxylase (ASPH, HAAH), which has been shown to be beneficial in cases of hepatocellular carcinoma in mice. Lambda phage has also been of major importance in the study of specialized transduction.