T4 coliphage is a phage that infect coliform bacteria especially E.coli. T4 is a double stranded DNA phage, it has a contractile sheath and an unique base 5-hydroxyl methyl cytosine (5-HMC). It is the best known member of large virulent phages.
The lytic cycle of T4 phage involve following steps: ADSORPTION
PENETRATION
BIOSYNTHESIS
ASSEMBLY
RELEASE
1]. Adsorption :
- The lytic cycle begins when a bacteriophage comes in contact with a susceptible host cell by random collision.
- Phage possess an adsorption organs or anti-receptors and host cells possess receptors.
- Host cell surface components -Flagella, Pilli , Teichoic acids, Proteins, Carbohydrates , LPs and Lipopolysaccharides serves as receptors.
- Phage components such as tail fibers, tail proteins and spikes serves as adsorption organs or anti-receptors.
- Each phage has its specific receptor to which it adsorbs.
- Adsorption takes place only when the anti-receptor is chemically complementary to the receptor.
- T4 phage possess tail fibers that serves as an adsorption organ or anti-receptor.
- Normally, tail fibers are present in folded form around the tail.
- Whiskers hold the tail fibers in folded form.
- When phage come in contact with host, tail fibers unfold.
- Unfolding of fibers requires tryptophan & co-factors - Mg++ & Ca++.
- Thus, the phage and host binding is favoured by ionic environment.
- T4 host E.coli possess outer membrane protein C (OmpC) – lipopolysaccharide complex as receptor.
- Initial attachment occurs when tail fibers attach to the OmpC- lipopolysaccharide complex.
- Initial adsorption is weak and reversible.
- It becomes irreversible when tail pins attach to lipopolysaccharide.
2]. Penetration :
- Once attached, the bacteriophage injects DNA into the bacterium.
- Bacteria possess rigid cell wall and therefore the phages directly cannot penetrate into the bacterial cells.
- They inject only their nucleic acids inside the host cell.
- In the T-even phage, irreversible binding of the phage to host results in the contraction of the sheath and the hollow tail tube is inserted through host cell wall.
- Some phages have enzymes like lysozyme that digest the cell wall components of the bacterial cell.
- The penetration of T4 phage DNA occurs when -
- There is irreversible attachment of phage to host cell,
- Contraction of sheath, pushing tail tube through cell envelope
- Injection of DNA into cell like injection of vaccine/drug by a syringe
3]. Biosynthesis :
Biosynthesis divided into three steps:- Formation of immediate early and delayed early protein
- Replication of phage genome
- Formation of late proteins
i) Formation of immediate early and delayed early protein :
- Part of phage DNA is immediately transcribed by host RNA polymerase to form immediate early m-RNAS.
- These early m-RNA translate to following enzyme proteins -
- a) Nucleases - Breaks down host DNA & make nucleotides available for its own synthesis.
- b) α-subunit modifying enzyme - modifies α-subunit of host RNA polymerase.
- Modified host RNA polymerase transcribes part of viral genome to delayed early m-RNAS.
- a) Phage enzymes that produce 5-hydroxyl methyl cytosine (5-HMC), a unique base in phage DNA
- b) Polymerases and ligases - that play role in phage DNA replication and recombination.
- c) Glucosylation enzyme-adds glucose to HMC & protects phage DNA from host restriction endonuclease
- d) σ-subunit modifying enzyme - modifies σ-factor of RNA polymerase so that is transcribes late mRNAs.
ii) Replication of Phage Genome :
Two modes have been proposed for the replication of T4 phage DNA.By bi-directional mode - at early stage
By rolling circle mode - at later stage
- Initial replication is bi-directional and semi-discontinuous.
- Leading strand is synthesized continuously and lagging strand is synthesized discontinuously leading to the formation of eye structure.
- Bi-directional replication is initiated at several origins along the DNA and is catalyzed by phage coded enzymes.
- In the rolling circle mode of replication, a cut is made in one of the DNA strands by a specific endonuclease and 3'end is made free.
- DNA polymerase extends the free 3'OH end by adding complementary bases.
- Intact strand serves as template for addition of complementary bases.
- Due to extension of 3'OH end, the 5'end is displaced.
- Displaced strand is synthesized discontinuously by adding Okazaki fragments.
- This mechanism produces multi-genome length molecules.
- Such molecules are referred to as concatemers.
- The concatemers are later cleaved to head sized molecules by headful cutting mechanism.
- Soon after the replication of phage DNA, transcription of late m-RNAs occur.
- These late m-RNAs translate to different proteins.
- These proteins include the structural proteins.
- They are proteins involved in phage assembly and an enzyme lysozyme that degrades the peptidoglycan layer of bacterial cell wall.
- For example - head (capsid) proteins, tail tube protein, sheath proteins, collar, whiskers, base plate, tail fiber, tail pins, lysozyme etc.
4. Assembly of Phages :
- Assembly of new phage particles begins after accumulation of structural proteins and nucleic acid molecules in the cell.
- Process of assembling phage particles is known as known as maturation.
- There are four different pathways that lead to the formation of phage particles.
- These include base plate, tail tube & tail sheath, tail fibers and head.
- About 50 genes take part in the morphogenesis of T4 phage.
- Subunits of base plate assemble to form a base plate.
- Then tail tube and sheath subunits polymerize on base plate to form mature tail.
- The subunits of head assemble together to form prohead and then DNA is inserted in the prohead to form complete head.
5. Release :
- The release of newly synthesized phages occurs by sudden explosion or bursting (lysis) of bacterial cell.
- Lysis begins after about 22 minutes.
- One of the gene products involved in the process include lysozyme.
- It cleaves glycosidic bonds in the peptidoglycan making the cell wall susceptible to the rupture.
- There is another protein termed as holin that make holes in the cell membrane and makes the way for lysozyme action.