Phage lambda

Bacteriophage λ is the archetype of the lambdoidphages and belongs to the lambda-like phage subclass of the siphoviridae. Lambda is a temperate phage; it can grow either lytically or lysogenically. This feature has made λ especially important to the field of molecular biology. Discoveries based on λ include the physical nature of and site-specific recombination, DNA ligase, SDS-PAGE, cloning, and retroregulation. The λ strain used most often in labs, although called wt, is λPaPa, named after the parental strains from Pasedena California and the Institut Pasteur in Paris.

Morphology

The λ phage has an icosahedral head (with T=7) about 50-60 nm in diameter, and a flexible, long noncontractile tail about 150nm in length. The head is formed from the proteins E, D, B, W, FII, B*, X1, and X2. The head is the capsid, and contains a single double-stranded DNA molecule about 48 kb in length; by weight the genome is about 32x10⁶ Da and comprises about half of the phage's weight. The tail consists of a 135nm hollow tube finished with a 15nm conical cap. The inside diameter of the tail is about 3nm, with an outside diameter of 9-18nm due to knob-like structures that make the tail appear rough. The wt λ does not have tail fibers, although these may be present on certain strains.

Genome Organization

The 48,502bp dsDNA is linear when packaged, and recircularizes using host ligase at the cos (cohesive end site) once injected into the host due to complementary 5' ends.. The information strand, rightmost (Rz) to leftmost (nu1), contains regions for lysis, late control, a non-essential b region, DNA replication, repression (cII,cro, cI), antitermination (cIII), recombination, excision & integration, another non-essential region nin, head genes and then tail genes. The att (attachment) site, containing xis and int, is between the recombination and the rightmost non-essential regions. The b region comprises about 18% of the genome, while the nin (N independence) region is much smaller. The cos region consists of the cosQ, cosN, and cosB subsites, and is flanked by nu1.

As a prophage, the λ genome is inserted between the host gal genes and bio genes genes at the att sites.

Life Cycle

Lytic Cycle

The lytic cycle produces progeny phage particles and results in the death of the host bacteria.

Adsorbtion and Injection

The J protein, located at the tip of the tail, attaches to the host maltoporin lamB, which is displayed on the outer membrane. The original isolate was able to infect hosts using ompF as well as lamB. The dsDNA is stored in the phage head as a linear strand, and is fed through the tail after a conformational change caused by the interaction of the phage tail J protein with the host protein lamB . The genome enters "right" end first and recircularizes immediately upon entry into the host due to complementary 12nt long sticky ends. The sticky ends are each neighbored by packing signals, and the entire site is termed cohesive end site, or cos. Host DNA ligase is needed to covalently seal the ends together, and host gyrase is required to catalyze the negative supercoiling of the relaxed genome.

Early Gene Expression

Transcripton requires host RNA polymerase and occurs almost immediately from p_R (for promoter right) to the t_R1 terminator, producing the cro gene product, and simultaneously from p_L to t_L1, resulting in production of N. Neither termination is completely efficient; about 20% of transcripts continue from p_L through t_L1 to t_L2 and result in cIII. Transcription from p_R through t_R1 to t_R2, which occurs with about half the transcripts, produces cII, O and P. Gam is an early gene product which inhibits host exonuclease V (recB/recC/recD).

Middle Gene Expression

The middle phase of gene expression occurs about 3-9 minutes after host infection. The early gene product N behaves like an antiterminator by working in tandem with host nusA and causes RNA polymerase to read past t_L1 and t_R1; as well as higher amounts of N, cIII and products needed for recombination and integration from p_L, the phage produce cro, cII, O,and P from p_R. Transcription to t_R3, which is unaffected by by the action of N, produces another antiterminator, Q. Another early gene product, cro, works against N by binding at he repressing transcription from p_L and, at a slightly higher concentration, from p_R, thereby blocking transcription of all early and middle genes.

DNA Replication

Both O and P, as well as several host enzymes, are needed for replication.There are two phases of replication in λ. Replication first proceeds via θ (theta) replication, which begins during the middle stages of transcription. θ replication originates from the O/cII gene region once host gyrase has supercoiled the λ DNA; two daughters, both circular, are produced by each full round of θ replication.

Replication proceeds in the late stages, about 15 minutes post infection, via rolling circle (σ) replication when entering the lytic cycle, and the resulting concatemers are cut on the (+) strand by phage-encoded endonuclease.

Late Gene Expression

Late genes are expressed from about 9 minutes post-phage entry. After about 15 minutes, accumulation of the repressor cro allows it to prevent transcription of the early genes (from p_Rand p_L) by binding to the operators o_R and o_L. The production of Q allows for the transcript from p_R' to proceed through p_R', resulting in higher levels of S and R. The production of cII and cIII activate p_I (integrase) and p_RE (repression establishment).

The antiterminator Q allows transcription past t_R4.

Packaging

Proteins B, C, and Nu3 form the capsid precursors, which are aided by host groEL and groES to combine with E and form an immature capsid, petit λ. The DNA formed by rolling circle replication remains as concatomers until terminase cleaves the cos site and one end of the DNA is packed into the capsid. Packaging requires some host enzymes and the phage proteins nu1, A, FI and D. The proteins E and C combined and are reformed into X1 and X2, which make a mature capsid once B* (modified B) is added and Nu3 is degraded. D protein is added to the capsid and standardizes the size of the head; the λ DNA is then inserted into the head, "left" end first, until the terminase cuts the DNA at the next cos site and W and FII proteins stabilize the capsid.

Tails are formed independently of the head, and construction is initiated at the far end of the structure from the proteins in the order J, I, L, K, H, G, M, V, U, H, and Z. Only the J protein interacts with the host cell; the tube consists of 32 hexameric rings of V joined to the head by U. The tail component attaches spontaneously to mature capsids by proteins W and FII.

Lysis

Proteins R and S are used to degrade the bacterial membranes. S creates a hole in the inner membrane; R is released through this hole into the periplasm and degrades the cell wall. The influx of water renders the cell unable to maintain turgor and the cell explodes. About 100 phage are released per host, and infection to release takes about 35 minutes (40 min at 37˚C).

Lysogenic State

The lysogenic lifecycle does not result in the production of a large number of progeny phage, nor is the host cell destroyed. Instead, recombination of the λ DNA with the host genome creates a prophage which is replicated as part of the host chromosome with each cell division. The single recombination event occurs between specific sites: the attP (for attachment phage) on the λ DNA and the attB (for attachment bacteria) loci. The genes that cause lysis must be repressed in order for a lysogen to integrate. Most of the phage genes are silenced, as very few genes are required to keep the phage DNA integrated. Prophage and descendant prophage are capable of producing phage when the λ DNA is induced to excise from the host DNA.

Lysis-Lysogeny Switch

The transcriptional repressors cI and cro can repress each other and compete for the operator o_R. These two proteins also regulate cII, which is an unstable product and is rapidly degraded unless cIII is present.

Expression of cI represses the lytic cycle. The λ repressor, which binds to o_R and o_L, is the result of p_RE and can shut off production of O, P, and Q if produced in high quantities. The repression of p_R and p_L also prevents transcription of cII and cIII. The integrase produced from p_I allows for induction of the phage DNA into the host DNA.

Prophage Induction

When cII is bound to its promoter p_I (for integration), production of int allows for the integration of λ DNA into the host genome.

Since the phage genome is circular, only one recombination event is needed and all of the phage genes can be retained. The attP (for attachment phage) site can recombine with the host DNA attB (bacteria) due to a 15bp region of homology and the adjacent non-homologous sequences.

Although only int and a few host enzymes are needed for integration, excision requires both cII and xis. The xis product is necessary for the induction process, where repression of the vegetative (lytic) cycle is reversed. Induction can be stimulated by stressing the host with enviromental conditions or antibiotics.

See also

• Lambda Gene List
• Lambda-like phage
• Enterobacteriophage lambda at the NCBI taxonomy browser
• wikipedia: Lambda_phage
• ICTVdb entry
• Siphoviridae at MicrobeWiki
• Phage lambda on UniProt

References

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