Lytic cycle: Multiplication of T4 Bacteriophage

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

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 -
  1. There is irreversible attachment of phage to host cell,
  2. Contraction of sheath, pushing tail tube through cell envelope
  3. Injection of DNA into cell like injection of vaccine/drug by a syringe

3]. Biosynthesis :

Biosynthesis divided into three steps:
  1. Formation of immediate early and delayed early protein
  2. Replication of phage genome
  3. 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.
Delayed early mRNAs are translated to following enzymes-

  •  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.
III] Formation of late proteins 
  • 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.

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