Bacteriophage Lambda: A Temperate Bacteriophage

Unlike bacteriophage T4, temperate bacteriophages, such as phage lambda (family Siphoviridae, species Enterobacteria phage lambda), can enter either the lytic or lysogenic cycle upon infecting a host cell.

If a temperate virus enters the lysogenic cycle, its dsDNA genome often is integrated into the host’s chromosome, where it resides as a prophage until conditions for induction occur (see figure 6.16). Upon induction, the viral genome is excised from the host genome and the lytic cycle is initiated

Phage lambda virion
Bacteriophage Lambda (l).
Lytic and lysogenic infections are common for bacterial and archaeal cells

How does a temperate phage “decide” which cycle to follow? The process by which phages make this decision is best illustrated by bacteriophage lambda (l), which infects E. coli. As shown in figure, l has an icosahedral head 55 nm in diameter and a noncontractile tail with a thin tail fiber at its end.

Its DNA genome is a linear molecule with cohesive ends—singlestranded stretches, 12 nucleotides long, that are complementary to each other and can base-pair. Like most bacteriophages, l attaches to its host and then releases its genome into the cytoplasm, leaving the capsid outside.

Once inside the cell, the linear genome is circularized when the two cohesive ends base-pair with each other; the breaks in the strands are sealed by the host cell’s DNA ligase (figure 27.9). The l genome has been carefully mapped, and over 40 genes have been identified (figure).

Most genes are clustered according to their function, with separate groups involved in head synthesis, tail synthesis, lysogeny, DNA replication, and cell lysis.

This organization is important because once the genome is circularized, a cascade of regulatory events occurs that determine if the phage pursues a lytic cycle or establishes

Lambda Phage DNA.
Lambda Phage DNA.

lysogeny. Regulation of appropriate genes is facilitated by clustering and coordinated transcription from the same promoters. The cascade of events leading to either lysogeny or the lytic cycle serves as a model for complex regulatory processes.

It involves the action of several regulatory proteins that function as transcriptional repressors or activators or both, proteins that regulate transcription termination, and antisense RNA molecules (figur). The protein cII is an activator protein that plays a pivotal role in determining if l will establish lysogeny or follow a lytic pathway.

If the cII protein reaches high enough levels early in the infection, lysogeny will occur; if it does not reach a critical level, the lytic cycle will occur.

The Genome of Phage Lamda
The Genome of Phage Lamda

Transcription of the l genome is catalyzed by the host cell’s DNA-dependent RNA polymerase (an RNA polymerase that uses DNA as the template for RNA synthesis), and the cII protein is synthesized relatively early in the infection.

If the cII protein levels are high enough, it will increase transcription of the int gene, which encodes the enzyme integrase. Integrase catalyzes integration of the l genome into the host cell’s chromosome, thus establishing lysogeny.

The cII protein also increases transcription of the cI gene. This gene encodes a regulatory protein that is often called the l repressor because it represses the transcription of all genes (except its own). This repression maintains the lysogenic state. As just noted, the cI protein (l repressor) allows transcription of its own gene.

This is because cI functions as an activator protein when it binds to the PRM promoter from which the cI gene can be transcribed (table). Integration of the l genome into the host chromosome takes place at a site in the host chromosome called the attachment site (att).

A corresponding site is present on the phage genome, and the l integrase catalyzes site-specific recombination between the two sites.

The bacterial site is located between the galactose (gal) and biotin (bio) operons, and as a result of integration, the circular l genome becomes a linear stretch of DNA located between these two host operons (see figure). The prophage can remain integrated indefinitely, being replicated as the bacterial genome is replicated.

Because the cII protein is made early in the infection, it might seem that it would accumulate quickly and ensure that lysogeny occurs. However, cII is degraded by a host enzyme (HflB) unless it is protected by a viral protein called cIII. The cIII protein is synthesized at the same time as cII, and as long as its levels remain high enough, cII will be protected.

However, if cII is not protected sufficiently from the host degradative enzyme, the level of a protein called Cro (product of the cro gene) will increase. Cro protein is both a repressor protein and an activator protein.

It inhibits transcription of the cIII and cI genes, further decreasing the amount of cII and l repressor. However, it increases its own synthesis as well as the synthesis of another regulatory protein called Q.

When Q protein accumulates at a high enough level, it activates transcription of genes required for the lytic cycle. When that occurs, the infection process is committed to the lytic cycle. Ultimately the host is lysed and new virions are released.

We have now considered the regulatory processes that dictate whether lysogeny is established or the lytic cycle is pursued. However, how does induction reverse lysogeny? Induction usually occurs in response to environmental factors such as ultraviolet light or chemical mutagens that damage DNA.

The Decision Making Process for Establishing Lysogeny or the Lytic Pathway.
The Decision-Making Process for Establishing Lysogeny or the Lytic Pathway.
The initial transcripts are synthesized by the host RNA polymerase. These encode the N protein and the Cro protein. The N protein is an antiterminator that allows transcription to proceed past the terminator sequences tL, tR1, and tR2. This allows transcription of other regulatory genes, as well as the xis and int genes. The latter genes encode the enzymes excisionase and integrase, respectively. The left side of the figure illustrates what occurs if lysogeny is established. The right side of the figure shows the lytic pathway.

This damage alters the activity of the host cell’s RecA protein. As we describe in chapter 16, RecA plays important roles in recombination and DNA repair. When activated by DNA damage, RecA interacts with l repressor, causing the repressor to cleave itself.

As more and more l repressor proteins destroy themselves, transcription of the cI gene is decreased, further lowering the amount of l repressor in the cell. Eventually the level becomes so low that transcription of the xis, int, and cro genes begins. The xis gene encodes the protein excisionase. It binds integrase, causing

Functions of Lambda Promoters and Operators

it to reverse the integration process, and the prophage is freed from the host chromosome. As l repressor levels continue to decline, the Cro protein levels increase. Eventually, synthesis of l repressor is completely blocked, protein Q levels become high, and the lytic cycle proceeds to completion.

Our attention has been on l phage, but there are many other temperate phages. Most, like l, exist as integrated prophages in the lysogen. However, not all temperate phages integrate into the host chromosome at specific sites.

Bacteriophage Mu integrates randomly into the genome. It then expresses a repressor protein that inhibits lytic growth. Furthermore, integration is not an absolute requirement for lysogeny. The E. coli phage P1 is similar to l in that it circularizes after infection and begins to manufacture repressor.

However, it remains as an independent circular DNA molecule in the lysogen and is replicated at the same time as the host chromosome. When E. coli divides, P1 DNA is apportioned between the daughter cells so that all lysogens contain one or two copies of the phage genome

Key Concepts

Single-Stranded DNA Viruses Use a Double Stranded Intermediate in Their Life Cycles

■  ϕX174 is an example of a ssDNA bacteriophage. Its replication involves the formation of a dsDNA replicative form (RF) (figure).

■ fd phage is filamentous phage that upon infection is continuously released by the host without causing lysis.

■ Parvoviruses cause a spectrum of diseases in a wide variety of animals. The parvovirus genome is replicated by host DNA polymerase in the host cell’s nucleus using a process that is similar to rolling-circle replication (figure).

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