Lambda phage


Lambda phage is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia 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, a tail, and tail fibers. 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.

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 and requires 7 more for assembly. 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.
  1. 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 of E. coli, a porin molecule, which is part of the maltose operon.
  2. The linear phage genome is injected through the outer membrane.
  3. The DNA passes through the mannose permease complex in the inner membrane 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.
  4. Host DNA gyrase puts negative supercoils in the circular chromosome, causing A-T-rich regions to unwind and drive transcription.
  5. 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.
  6. Cro binds to OR3, preventing access to the PRM promoter, preventing expression of the cI gene. N binds to the two Nut sites, one in the N gene in the PL reading frame, and one in the cro gene in the PR reading frame.
  7. 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 include the N and cro genes along with cII and cIII genes, and xis, int, O, P and Q genes discussed later.
  8. The cIII protein acts to protect the cII protein from proteolysis by FtsH by acting as a competitive inhibitor. This inhibition can induce a bacteriostatic state, which favours lysogeny. cIII also directly stabilises the cII protein.
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 are known to favor lysogeny.

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.
  1. The boxB RNA sequences are located close to the 5' end of the pL and pR transcripts. When transcribed, each sequence forms a hairpin loop structure that the N protein can bind to.
  2. N protein binds to boxB in each transcript, and contacts the transcribing RNA polymerase via RNA looping. The N-RNAP complex is stabilized by subsequent binding of several host Nus proteins.
  3. The entire complex continues transcription, and can skip through termination sequences.

    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.
  1. The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome. Cro dominates the repressor site, repressing synthesis from the PRM promoter .
  2. The O and P proteins initiate replication of the phage chromosome.
  3. Q, another antiterminator, binds to Qut sites.
  4. Transcription from the PR' promoter can now extend to produce mRNA for the lysis and the head and tail proteins.
  5. Structural proteins and phage genomes self-assemble into new phage particles.
  6. 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

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.
Pr promoter has been found to activate the origin in the use of rightward transcription, but the whole picture of this is still somewhat misunderstood. Given there are some caveats to this, for instance this process is different for other phages such as N15 phage, which may encode for DNA polymerase. Another example is the P22 phage may replace the p gene, which encodes for an essential replication protein for something that is capable of encoding for a DnaB helices.

Lytic replication

  1. For the first few replication cycles, the lambda genome undergoes θ replication.
  2. This is initiated at the ori site located in the O gene. O protein binds the ori site, and P protein binds the DnaB subunit of the host replication machinery as well as binding O. This effectively commandeers the host DNA polymerase.
  3. Soon, the phage switches to a rolling circle replication similar to that used by phage M13. The DNA is nicked and the 3' end serves as a primer. Note that this does not release single copies of the phage genome but rather one long molecule with many copies of the genome: a concatemer.
  4. These concatemers are cleaved at their cos sites as they are packaged. Packaging cannot occur from circular phage DNA, only from concatomeric DNA.