Lambda phage

Enterobacteria phage λ (lambda phage) is a temperate bacteriophage that infects Escherichia coli.

Lambda phage is a virus particle consisting of a head, containing 48,514 base-pairs of double-stranded linear DNA as its genetic material, 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 where it replicates its DNA, degrades the host DNA and hijacks the cell's replication, transcription and translation mechanisms to produce as many phage particles as cell resources allow. When cell resources are depleted, the phage will lyse (break open) the host cell, releasing the new phage particles. 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. These 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.

Lambda phage was discovered by Esther Lederberg in 1950.^[1] It has been used heavily as a model organism, and has been a rich source for useful tools in molecular biology. 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.

In the following page, we will write genes in italics and their associated proteins in Roman. For instance, cI refers to the gene, while cI is the resulting protein encoded by that gene.

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Lifecycle

Infection

1. Bacteriophage Lambda binds to the target E. coli cell, the J protein in the tail tip interacting with the lamB gene product of E. coli, a porin molecule which is part of the maltose operon.
2. The linear phage genome is injected past the cell outer membrane.
3. The DNA passes through a separate sugar transport protein (ptsG) in the inner membrane, and immediately circularises using the cos sites, 12-base G-C rich cohesive "sticky ends". The single-stranded nicks are ligated by host DNA ligase.
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 P_L, P_R and P_R' promoters producing the 'immediate early' transcripts. Initially these express the N and cro genes, producing N, Cro and a short inactive protein.
6. Cro binds to OR3 preventing access to the P_RM promoter preventing expression of the cI gene. N binds to the two Nut (N utilisation) sites, one in the N gene in the P_L reading frame, and one in the cro gene in the P_R reading frame.
7. The N protein is an antiterminator, and functions to extend the reading frames that it is bound to. When RNA polymerase transcribes these regions, it recruits the N and forms a complex with several host Nus proteins. This complex 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, OP and Q genes discussed later.
8. The cIII protein acts to protect the cII protein from proteolysis by FtsH (a membrane-bound essential E. coli protease) by acting as a competitive inhibitor. This inhibition can induce a bacteriostatic state, which favours lysogeny. cIII also directly stabilises the cII protein^[2]. 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).

Lytic Lifestyle

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 (OP). Cro dominates the repressor site (see "Repressor"), repressing synthesis from the P_RM promoter.
2. The O and P proteins initiate replication of the phage chromosome (see "Lytic Replication").
3. Q, another antiterminator, binds to Qut sites.
4. Transcription from the P_R' 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 lysis genes R and S, cause cell lysis at high enough concentrations. S is a holin which makes holes in the membrane. R is an endolysin which cleaves the cell wall. Around 100 new phage are released.

Rightward Transcription

Rightward transcription expresses the O, P and Q genes. O and P are responsible for initiating replication, and Q is another antiterminator which allows the expression of head, tail and lysis genes from P_R’.

Lytic Replication

1. For the first few replication cycles, the lambda genome undergoes θ replication (circle-to-circle).
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 type of replication similar to that used by phage M13. The DNA is nicked and the 3’ end serves as a primer. Notably, this doesn’t 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.

Q Antitermination

1. 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.
2. The Qut site is very close to the P_R’ promoter, close enough that the σ factor has not been released from the RNA polymerase holoenzyme. The Qut site resembles the -10 Pribnow box, causing the holoenzyme to pause.
3. Q protein then binds and displaces part of the σ factor and transcription re-initiates.
4. The head and tail proteins are transcribed and self-assemble.

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 which are vital to lysogeny.

xis and int regulation of insertion and excision

1. 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.
2. The mRNA from the P_L promoter forms a stable secondary structure with a bobby pin loop in the sib section of the mRNA. This targets the 3' (sib) end of the mRNA for RNAaseIII degradation, which results in a lower effective concentration of int mRNA than xis mRNA (as the int cistron is nearer to the sib sequence than the xis cistron is to the sib sequence), so a higher concentrations of xis than int is observed.
3. Higher concentrations of xis than int result in no insertion or excision of phage genomes, the evolutionarily favoured action - leaving any pre-insterted phage genomes inserted (so reducing competition) and preventing the insertion of the phage genome into the genome of a doomed host.

Lysogenic (or Lysenogenic) Lifestyle

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.

1. The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome.
2. The stablized cII acts to promote transcription from the P_RE, P_I and P_antiq promoters.
3. The P_antiq promoter produces antisense mRNA to the Q gene message of the P_R promoter transcript, thereby switching off Q production. The P_RE promoter produces antisense mRNA to the cro section of the P_R promoter transcript, turning down cro production, and has a transcript of the cI gene. This is expressed, turning on cI repressor production. The P_I 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").
4. No Q results in no extension of the P_R' 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 P_R promoter, turning off cro and other early gene expression. cI also binds to the P_L promoter, turning off transcription there too.
5. Lack of cro leaves the OR3 site unbound, so transcription from the P_RM promoter may occur, maintaining levels of cI.
6. Lack of transcription from the P_L and P_R promoters leads to no further production of cII and cIII.
7. As cII and cIII concentrations decrease, transcription from the P_antiq, P_RE and P_I stop being promoted since they are no longer needed.
8. Only the P_RM and P_R' 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.

Prophage Integration

The integration of phage λ takes place at a special attachment sites in the bacterial and phage genomes, called att^λ. 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.

Maintenance of Lysogeny

• Lysogeny is maintained solely by cI. cI represses transcription from P_L and P_R while upregulating and controlling its own expression from P_RM. It is therefore the only protein expressed by lysogenic phage.
• This is coordinated by the P_L and P_R operators. Both operators have three binding sites for cI: OL1, OL2, and OL3 for P_L, and OR1, OR2 and OR3 for P_R.
• cI binds most favorably to OR1; binding here inhibits transcription from P_R. As cI easily dimerises, the binding of cI to OR1 greatly increases the affinity of the binding of cI to OR2, and this happens almost immediately after OR1 binding. This activates transcription in the other direction from P_RM, as the N terminal domain of cI on OR2 tightens the binding of RNA polymerase to P_RM and hence cI stimulates its own transcription. When it is present at a much higher concentration, it also binds to OR3, inhibiting transcription from P_RM, thus regulating its own levels in a negative feedback loop.
• cI binding to the P_L 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 P_L and P_R promoters.

Induction

1. The host cell, containing a dormant phage genome, experiences DNA damage due to a high stress environment, and starts to undergo the SOS response.
2. RecA (a cellular protein) detects DNA damage and becomes activated. It is now RecA*, a highly specific co-protease.
3. Normally RecA* binds LexA (a transcription repressor), activating LexA auto-protease activity,which destroys LexA repressor allowing production of DNA repair proteins. In lysogenic cells this response is hijacked, and RecA* stimulates cI autocleavage.
4. Cleaved cI can no longer dimerise, and loses its affinity for DNA binding.
5. The P_R and P_L promoters are no longer repressed and switch on, and the cell returns to the lytic sequence of expression events (note that cII is not stable in cells undergoing the SOS response). There is however one notable difference.

Control of phage genome excision in induction

1. The phage genome is still inserted in the host genome and needs excision for DNA replication to occur. The sib section beyond the normal P_L promoter transcript is, however, no longer included in this reading frame (see diagram).
2. No sib domain on the P_L promoter mRNA results in no hairpin loop on the 3' end, and the transcript is no longer targeted for RNAaseIII degradation.
3. The new intact transcript has one copy of both xis and int, so approximately equal concentrations of xis and int proteins are produced.
4. Equal concentrations of xis and int result in the excision of the inserted genome from the host genome for replication and later phage production.

Genome Structure

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 pRM promoter (preventing cI production). At high concentrations downregulates its own production through OR2 and OR1 binding. No cooperative binding (c.f. below for cI binding)

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). At high concentrations downregulates its own production through OR3 binding. Binding of cI at OR1 stimulates an almost simultaneous cI binding to OR2 via cooperative binding (via cI C terminal domain interactions) N terminal domain of cI on OR2 tightens the binding of RNA polymerase to pRM and hence stimulate its own transcription. Repressor also inhibits transcription from the pL promoter. Susceptible to cleavage by RecA* in cells undergoing the SOS response.

cII; (Clear 2) Transcription activator. Activates transcription from the pAQ, pRE and pI promoters. Low stability 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 RNA polymerase cofactor, binds RNA (at Nut sites) and transfers onto the nascent RNApol that just transcribed the nut site. This RNApol modification 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 RNApol cofactor, binds DNA (at Qut sites) and transfers onto the initiating RNApol. This RNApol modification alters its recognition of termination sequences, so normal ones are ignored; special Q termination sequences some 20,000 bp away are effective.

xis; (eXcISion) excisionase and Int protein regulator, manages excision and insertion of phage genome into the host's genome.

int; (INTegration) Int 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 [Shown on diagram as head and tail, 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]; structural proteins, self assemble with the phage genome into daughter phage particles.

S, R [Shown on diagram as lysis. The order shown here is as found on the genome, reading in a clockwise direction]; cause the host cell to undergo lysis at high enough concentrations.

OP [Shown on diagram as O replication 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. 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.

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.^[3]

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 dimer also known as the cI protein. 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.

• In the absence of cI proteins, the cro gene may be transcribed.
• In the presence of cI proteins, only the cI gene may be transcribed.
• At high concentration of cI, transcriptions of both genes are repressed.

Lytic or Lysogenic?

The gene regulatory circuitry of phage λ is among the best-understood circuits at the mechanistic level. This circuitry involves several interesting regulatory behaviors. An infected cell undergoes a decision between two alternative pathways, the lytic and lysogenic pathways. If the latter is followed, the lysogenic state is established and maintained. While this state is highly stable, it can switch to the lytic pathway in the process of prophage induction, which occurs when the host SOS response is triggered by DNA damage.^[4].

Simplistically, in cells with abundant 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.

References

Significant papers

• Esther Lederberg, "Lysogenicity in Eescherichia coli strain K-12", Microbial Genetics Bulletin, v.1, pp. 5-8 (Jan. 1950); followed by "Lysogenicity in E. coli K-12", Genetics, v.36, p. 560 (1951) (abstract) (discovery reported).
• Mark Ptashne and Nancy Hopkins, "The Operators Controlled by the Lambda Phage Repressor", PNAS, v.60, n.4, pp. 1282-1287 (1968).
• Barbara J. Meyer, Dennis G. Kleid, and Mark Ptashne, "Lambda Repressor Turns Off Transcription of Its Own Gene", PNAS, v.72, n.12, pp. 4785-4789 (Dec. 1975).

Basic overviews

• Brussow, H. and Hendrix, R.W. 2002 "Small is beautiful.", Cell, 108, 13-16
• Dodd, J.B. Shearwin, K.E. and Egan, J.B. 2005 "Revisited gene regulation in phage lambda.", Curr Opin Genet Dev, 15, 145-152
• Friedman, D.I. and Court, D.L. 2001 "Bacteriophage lambda; alive and well and still doing its thing.", Current Opinion in Microbiology, 4, 201-207
• Gottesman, M. and Weisberg, R.A. 2004 "Little lambda - who made thee?", Micro and Mol Biol Revs, 68, 796-813 (available online at Microbiology and Molecular Biology Reviews, American Society for Microbiology)
• Hendrix, R.W. et al 1999 "All the world's a phage.", Proc Nat Acad Sc USA, 96, 2192-2197
• Kitano, R. 2002 "Systems biology: a brief overview.", Science, 295, 1662-1664
• Ptashne, M. "A Genetic Switch: Phage Lambda Revisited", 3rd edition 2003
• Ptashne, M. 2005 "Regulation of transcription: from lambda to eukaryotes.", Trends in Biochem Sci, 30, 275-279
• Snyder, L. and Champness, W. "Molecular Genetics of Bacteria", 3rd edition 2007 (Contains an informative and well illustrated overview of bacteriophage lambda)