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This lectures deals about
- Temperate phages and their types
- Life cycle of four important temperate phages
Temperate Phages
Temperate Phages
Some DNA phages can establish a different relationship with their host. After adsorption and penetration, the viral genome does not take control of its host and destroy it while producing new phages. Instead, the viral genome remains within the host cell and replicates with the bacterial genome to generate a clone of infected cells that may grow and divide for long periods while appearing perfectly normal. The infected host bacterial cell will have slight reduction in their growth rate. This relationship between the phage and its host is called lysogeny.
Bacteria having the potential to produce phage particles under some conditions are said to be lysogens or lysogenic, and phages able to enter into this relationship are temperate phages.
The latent form of the virus genome that remains within the host but does not destroy it is called the prophage. The prophage usually is integrated into the bacterial genome but sometimes exists independently.
Induction is the process by which phage reproduction is initiated in a lysogenized culture.
Lambda phage
Lambda phage
The lambda phage, family Siphoviridae, that uses the K12 strain of E. coli as its host is the best-understood temperate phage and will serve as our example of lysogeny. Lambda is a double stranded DNA phage possessing an icosahedral head 55 nm in diameter and a noncontractile tail with a thin tail fiber at its end. The DNA is a linear molecule with cohesive ends. The lambda genome has been carefully mapped, and over 40 genes have been located.
Replication
Lambda phage can replicate using a normal lytic cycle. Immediately after lambda DNA enters E. coli, it is converted to a covalent circle, and transcription by the host RNA polymerase is initiated. Under lytic cycle, Lambda DNA replication and virion assembly are similar to the same processes already described for the T4 phage.
lambda DNA is primarily synthesized by way of the rolling-circle mechanism that are finally cleaved to give complete genomes.
Rolling-circle DNA replication
Rolling-circle replication is special method of DNA replication by most of the temperate phages.
Rolling circle DNA replication is initiated by an initiator protein encoded by the bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at origin of replication. The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Displacement of the nicked strand is carried out by a host-encoded helicase. Continued DNA synthesis can produce multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series called a concatemer. These linear copies can be converted to double-stranded circular molecules through the following process:
- First, the initiator protein makes another nick to terminate synthesis of the first (leading) strand.
- RNA polymerase and DNA polymerase III then replicate the single-stranded origin DNA to make another double-stranded circle.
- DNA polymerase I removes the primer, replacing it with DNA, and
- DNA ligase joins the ends to make another molecule of double-stranded circular DNA.
Note: The mechanism involved in this video is applicable to phages too [Youtube video]
Lysogenic cycle of Lambda phage
The lysogenic cycle involves the lambda phage entering the cell, and instead of creating new virus particles, is characterized by integration of the bacteriophages nucleic acid into the host bacterium's genome. The newly integrated genetic material acts as an additional set of genes that can be replicated when the DNA of the host cell is replicated and the host cell divides. The virus is thus transmitted to daughter cells at each subsequent cell division, although the phages remain inert, causing no harm to the host cells.
Prophage is the term for a phage DNA in its dormant state, typically integrated into the host bacteria's chromosome, but also can include the rare cases where the phage exists as a stable plasmid within the host cell. The lysogenic bacterium multiples normally until some environmental induction, such as ionizing radiation or ultraviolet light threatens the bacterial cell and induces the prophage to initiate the lytic cycle. The prophage detached from the bacterial chromosome (called excision) and the prophage enters the lytic cycle.
Lytic and Lysogenic cycles of Lambda phage
How the integration and excision of Prophage occur in bacteria
Lysogenic cycle of Phage - Youtube video
Difference between lytic and lysogenic life cycle of phages (Youtube video)
M13 Phage
M13 Phage
- Filamentous phage composed of circular-single stranded DNA
- Genome length is 6407 nucleotides
- Capsid made up of 2700 capsomeres (major coat protein P8, capped with 5 copies of two different minor coat proteins P9, P6 and P3).
- The minor protein P3 attaches to the receptor at the tip of the host E. coli.
M13 Phage – diagram
M13 – TEM Photo
Life cycle of M13
The general stages to a viral life cycle are: infection, replication of the viral genome, assembly of new viral particles and then release of the progeny particles from the host. M13 phage uses a bacterial F pilus to infect E. coli, with the M13 P3 tip contacting the bacterial pilus. The phage genome is then transferred to the cytoplasm of the bacterial cell where resident proteins convert the single stranded DNA genome to a double stranded replicative form (known as RF). This DNA then serves as a template for expression of the phage genes and due to their expression, the structural proteins will be synthesized. The RF also serves as template and by rolling-circle method of DNA replication, the genome of M13 (single strand DNA) will be synthesized. Then the assembly of proteins and ssDNA (viral) forms the complete M13 and release from the E. coli without killing the host.
Detailed description of M13 phage (Youtube)
ΦX174 (PhiX174) Phage
ΦX174 (PhiX174) Phage
Size of the genome is 5386 nucleotides. It is single stranded and circularly modulated DNA. It is encoded with 10 genes but generates 11 proteins. It is a lytic phage.
Infection is through binding to host cell’s lipopolysaccharide, especially N-acetyl glucose amine component of the outer membrane. A distinctive phospholipase found in this region has a role in viral infectivity. DNA penetrates to the cell and as soon as the ssDNA enters into cytoplasm, it gets coated with host ssBs and immediately the DNA is rendered super coiled.
The super coiled SSDNA is replicated to produce ds RF DNA, which in turn produces ssDNA (+) forms, which in turn generates more RF forms. Ultimately 500 or more viral particles produced are released by cell lyses.
Packing proteins and ssDNA in ΦX174
Life cycle of ΦX174
Life cycle of PaiX174 (Youtube)
Mu Phage
Mu Phage
Mu phage is a temperate phage (belongs to Myoviridae) infecting Enterobacterial species. It uses DNA-based transposition to integrate its genome into the genome of the host cell that it is infecting. It can then use transposition to initiate its viral DNA replication. Once the viral DNA is inserted into the bacteria, the Mu transposase protein/enzyme in the cell recognizes the recombination sites at the ends of the viral DNA and binds to them, allowing the process of replicating the viral DNA or embedding it into the host genome.
Transposition: The process of movement of segment of DNA from one position to another through transposase enzyme is referred as Transposition and the gene responsible for the process is called as transposon. Through the transposition, Mu phages induce the mutations in the bacterial genome, hence referred as Mu phages.
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