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Bacteriophages Help (page 2)

By — McGraw-Hill Professional
Updated on Aug 23, 2011

Lytic Cycles

The first step in the life cycle of phage T4 (Fig. 11.1) involves the adsorption of a virion to a specific receptor site on the surface of the host cell. Any cell lacking this receptor would be immune to infection by T4. Following adsorption, T4 injects its DNA through its tail into the host cell. The empty phage capsid remains outside the bacterium as a ghost (so named because of the empty appearance of the head in electron micrographs). The filamentous phage, M13, is able to penetrate the cell wall and then has its nucleic acid released by host-cell enzymes that digest the coat proteins.

Bacteriophage Life Cycles

Once the naked phage DNA is inside the cell, different phages may use different strategies to produce progeny particles. Generally, however, the phage DNA initially is transcribed by the host's RNA polymerase into "early mRNAs." Later mRNAs may be synthesized by a phage RNA polymerase that was made from an early mRNA; or perhaps the bacterial RNA polymerase becomes modified to transcribe phage genes preferentially or exclusively. These mRNAs become translated into enzymatic, regulatory, and structural proteins. The regulatory proteins of the phage control the timing at which various phage genes become active. The structural proteins form heads, tails, and other protein parts of the complete phage particle as needed. The phage enzymes mediate replication of many copies of the phage genome, further transcription, and sometimes even the destruction of the host's DNA.

EXAMPLE 11.2 Phage T4 specifies the enzyme hydroxymethylase that modifies the cytosine bases in its own DNA to 5-hydroxymethylcytosine. Such modified bases are resistant to degradation by host-cell nucleases, making the phage more successful during infection.

Several different mechanisms are known for packaging phage DNAs into their protein coats. In E. coli phage T4, rolling-circle replication of its double-stranded DNA produces long, tandemly linked series (concatemers) of phage genomes. It is thought that the end of the concatemer enters the head, followed by enough DNA to fill the head. The concatemer is then cleaved at a nonspecific site. Since the DNA capacity of the head is greater than the length of one phage genome (monomer), the gene order will be different in each linear fragment cut from the concatemer. Terminal regions will be present twice within each monomer (terminally redundant). Since each phage monomer cut from a concatemer begins at a different gene sequence, they collectively form a cyclically permuted set (Fig. 11.2).

Bacteriophage Life Cycles

In phage lambda (λ), the circular genome is replicated early in the lytic cycle to increase the number of templates for transcription and further replication. Later in the cycle, rolling-circle replication provides the genomes for packaging into the heads of progeny phage. Lambda genomes are also cut from a concatemer, but unlike phage T4, the cuts are made at base-specific sequences known as cos sites (for cohesive site). Linear phage genomes always end with single-stranded termini because they are cut from the concatemer at the cos site by a sequence-specific terminase or Ter system. Ter-cutting requires that two cos sites or one cos site and a free cohesive end (1/2 cos) be present on a single concatemeric DNA molecule. A modified lambda genome that is 79–106% the length of a normal λ phage genome will still be cut by the Ter system and become packaged into phage heads. This is an important property of λ that makes it useful as a vector for genetic cloning.

After assembly of the phage capsids is completed, the lytic protein lysozyme ruptures the cell and releases the progeny phage in a typical burst size of 50–300 infective particles per cell. Most virulent phages follow the general lytic cycle outlined above.

EXAMPLE 11.3 E. coli phage M13 is filamentous and contains a circular, single-stranded DNA molecule. Among all known phages reproducing vegetatively, M13 is the only one that neither kills nor lyses its host cell. Infective progeny phage leave the cell by budding from its surface without causing cell damage. Upon infection, the entire phage particle penetrates the cell wall by being absorbed at the end of a sex (F) pilus. The entry of coat proteins into the cell is another feature unique to this phage. One genetically engineered strain of M13 (M13mp7) contains the promoter (lacP), operator (lacO), and β-galactosidase gene (lacZ) of the E. coli lactose operon. Insertion of a foreign DNA segment into lacZ inactivates the gene and no enzyme is produced. Lac bacterial cells infected with wild-type M13mp7 would be able to ferment lactose. On EMB agar, lactose-fermenting colonies would appear dark purple. Cells exposed to a M13mp7 phage carrying a foreign DNA insert in lacZ would be unable to ferment lactose; therefore, they grow into colorless colonies. Like lambda, phage M13 has been widely used as a cloning vehicle in genetic engineering.

EXAMPLE 11.4 The temperate phage Mu inserts its DNA obligatorily into its E. coli host chromosome during its lytic cycle. These insertions are at random, and they often inactivate host genes or regulatory sequences. Insertion always results in duplication of a terminal target sequence. Thus, Mu is a giant transposon (page 314) that has acquired phage functions enabling it to be packaged into phage coats and to escape its host by lysis. Transposition is obligatory during Mu DNA replication. Insertion of progeny Mu DNA occurs at various sites throughout the lytic cycle. Various host DNA sequences are always found at the termini of Mu DNA. However, only Mu DNA inserts; the duplicated terminal bacterial sequences are not inserted.

Lysogenic Cycles

There are two types of lysogenic cycles. In the most common type, typified by E. coli phage lambda (λ), the phage DNA becomes integrated into the host chromosome. In the other type, represented by E. coli phage P1, the phage DNA does not integrate into the host chromosome, but somehow replicates in synchrony with it as a plasmid. Both the integrated and plasmid forms of phage DNA are called prophage.

The establishment of an integrated lambda prophage occurs in four major steps:

  1. Linear phage DNA is injected into the host bacterial cell; the phage DNA is circularized by base pairing of its terminally redundant tails.
  2. Some early phage genes are transcribed to produce a few molecules of a repressor protein and an integrase enzyme. The repressor then turns off transcription of phage genes.
  3. The phage DNA is usually integrated or inserted at a specific site into the host chromosome as a prophage with the aid of integrase.
  4. The bacterium survives and multiplies; the prophage is replicated along with the host chromosome.

Two conditions favor the establishment of the lysogenic cycle of a temperate phage: (1) depletion of nutrients in the growth medium and (2) high multiplicity of infection (MOI)—i.e., many adsorbed phages per bacterium. Phage can carry out the lytic cycle only in cells that are actively metabolizing. When nutrients are depleted, bacteria degrade their own mRNAs and proteins before they become dormant. When nutrients become available to an uninfected dormant bacterium, it can again resume growth. A phage-infected cell that becomes dormant interrupts the lytic cycle, and usually loses the ability to produce phage. The cell dies. On the other hand, if the cell can become lysogenized (containing a prophage), both the phage and the bacteriumcan survive a dormant period, and the potential for production of phage by induction persists (Fig. 11.3).

Bacteriophage Life Cycles

If a lysogenic bacterium sustains damage to its DNA, it would be advantageous for the prophage to deintegrate from the bacterial chromosome, enter the lytic cycle, produce progeny phage, and leave that cell. When bacterial DNA is damaged, a protease (RecA protein) of the SOS repair mechanism is activated. This protease cleaves the lambda repressor that has kept the prophage in its inactive state. The prophage DNA becomes derepressed, an excisionase enzyme is synthesized, and the prophage deintegrates from the host chromosome to enter the lytic cycle. This is the process known as prophage induction. If ultraviolet radiation has damaged the host DNA, the ensuing prophage induction is termed UV induction. When a nonlysogenic Fbacterial cell receives prophage from a lysogenic Hfr donor, the recipient cell dies by induction of the lytic phage cycle. This form of prophage induction is termed zygotic induction.

In the lysogenic cycle of phage P1, the prophage is not integrated into the bacterial chromosome. Upon entry into the cell, P1 DNA circularizes and is repressed. It remains as a free, supercoiled, plasmid-like molecule, and replicates once with each cell division so that each daughter cell receives one copy of the prophage.

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