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LYTIC AND
LYSOGENIC CASCADE
Medhavi Vashisth, Anu Bala, Priya Sharma, Taruna Anand, Nitin Virmani, B.C. Bera,
Rajesh K. Vaid
National Centre for Veterinary Type Cultures, ICAR- National Research Centre on Equines, Hisar, India
Several types of independent genetic units in bacteria
integrated
Plasmid vs. episome = lytic vs. lysogenic phage
free form
free form
integrated
integrated
conjugation
lysis
Some plasmids are able to integrate into the chromosome and are
thus replicated with the chromosome. Such plasmids are called
episomes.
• After bacteriophages have reproduced within
the host cell, many of them are released when
the cell is destroyed by lysis.
• A phage life cycle that culminates with the
host cell bursting and releasing virions is
called a lytic cycle, and viruses that reproduce
solely in this way are called virulent viruses.
• Eg. T-even phages of E. coli (Myoviridae)
LYTIC CYCLE
One Step Growth Experiment
• 1939 by Max Delbrück and Emory Ellis
• The reproduction of a large phage population is
synchronized so that the molecular events occurring
during reproduction can be followed.
• A culture of susceptible bacteria such as E. coli is mixed
with bacteriophage particles, and the phages are
allowed a short interval to attach to their host cells.
• The number of infective phage particles released from
bacteria is subsequently determined at various
intervals by a plaque assay
• During the latent period, which immediately
follows phage addition, there is no release of
virions.
• Rise period (burst) when the host cells rapidly lyse
and release infective phages. Finally, a plateau is
reached and no more viruses are liberated.
• The total number of phages released can be used
to calculate the burst size, the number of viruses
produced per infected cell.
• The latent period is called the eclipse period
because the virions detectable before infection are
now concealed or eclipsed.
The major events-
• Adsorption to the host cell
• Viral penetration of the host cell
• Synthesis of viral nucleic acids and proteins
• Assembly of phage particles
• Release of virions
Adsorption and Penetration
• Not random
• Receptor mediated-
• Cell wall lipopolysaccharides, proteins,
teichoic acids, flagella, and pili.
• Tail fiber contacts the appropriate receptor
• Baseplate settles down on the surface
• Binding is due to electrostatic interactions and is influenced by pH and
ions such as Mg2+ and Ca2+
• Tail sheath becomes shorter and wider (24 to 12 rings)
• Core is pushed through the bacterial wall
• The baseplate has gp5 (lysozyme): penetration through peptidoglycan
layer
• Linear DNA is extruded from the head, through the tail tube, and into the
host cell
Synthesis and assembly
Early mRNAs –
• Synthesis of protein factors and enzymes to
takeover cell machinery.
• Virus-specific enzymes degrade host DNA to
nucleotides
• Halting host gene expression
DNA replication – several oris, bidirectional
Early genes transcription –
• By host RNA polymerases & σ70
• Virus mediated transfer of ADP-ribose from NAD
to one α-subunit of RNA polymerase.
• Host gene transcription is inhibited.
• Second α-subunit receives an ADP-ribosyl group -
turns off some of the early T4 genes except motA-
transcription of late genes
• One of the later genes encodes σgp55. This sigma
factor helps RNA polymerase bind to late
promoters and transcribe late genes.
• Restriction – glucosylated HMC & methylation
Late mRNAs-
Directs the synthesis of three kinds of
proteins:
(1) phage structural proteins
(2) proteins that help with phage assembly
without becoming part of the virion structure
(3) proteins involved in cell lysis and phage
release.
Release of the phage particles
• Two proteins are involved.
• One directs the synthesis of an enzyme that attacks peptidoglycan
in the host’s cell wall. It is sometimes called lysozyme.
• Another protein called holin creates holes in the E. coli plasma
membrane, enabling lysozyme to move from the cytoplasm to the
peptidoglycan.
Endolysins –
• Endo-β-N-acetylglucosaminidase
• N-acetylmuramidase (lysozyme)
• Endopeptidase
• N-acetylmuramoyl-L-alanine amidase
• ϒ-D-glutaminyl-L-lysine endopeptidase
Temperate phages that have two reproductive
options:
• they can reproduce lytically as do the virulent
phages,
• or they can remain within the host cell without
destroying it.
• Lysogeny- relationship between a temperate phage
and its host.
• Prophage- The form of the virus that remains within
its host.
• Lysogens (Lysogenic bacteria)- The infected bacteria
Two distinctive characteristics-
• These cannot be reinfected by the same virus
(immunity to superinfection)
• Under appropriate conditions they lyse and release
phage particles (Induction)
Lysogenic conversion
• This occurs when a temperate phage induces a
change in the phenotype of its host.
• Often involve alterations in surface characteristics of
the host. Eg. Salmonella and epsilon phage
• Give the host pathogenic properties. Eg.
Corynebactium diptheriae and phage β
Two advantages :
• It allows a virus to remain viable within a dormant
host.
• In such situations, a prophage would survive but
most virulent bacteriophages would not be
replicated as they require active cellular
biosynthetic machinery.
• When there are many more phages in an
environment than there are host cells, a situation
virologists refer to as a high multiplicity of
infection (MOI).
• In these conditions, lysogeny allows for the survival
of host cells so that the virus can continue to
reproduce.
• Illustrated by bacteriophage lambda.
• Lambda is a double-stranded DNA phage that infects the
K12 strain of E. coli.
• Its DNA genome is a linear molecule with cohesive
ends.
• 40 genes. Most genes are clustered according to their
function, with separate groups involved in head
synthesis, tail synthesis, lysogeny, DNAreplication, 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 lysogeny.
• Regulation of appropriate genes is facilitated by
clustering and coordinated transcription from the same
promoter.
• Two regulatory proteins –
• Lambda repressor (product of the cI gene) - lysogeny
• Cro protein (product of the cro gene) – lytic cycle
• If lambda repressor prevails, the production of Cro
protein is inhibited and lysogeny occurs.
• If the Cro protein prevails, the production of lambda
repressor is inhibited and the lytic cycle occurs.
• This is because the lambda repressor prevents
transcription of viral genes, while Cro does just the
opposite: it ensures viral gene expression.
Lambda Repressor
• 236 aa long and folds into a dumbbell shape with globular
domains at each end.
• One domain binds DNA while the other binds another
lambda repressor molecule to form a dimer.
• Lambda repressor binds two operator sites OL and OR,
thereby blocking transcription of most viral genes.
• When bound at OL, it represses transcription from the
promoter PL. Likewise, when bound at OR it represses
transcription in the rightward direction from PR .
• However, it also activates transcription in the leftward
direction from the cI promoter PRM
(cI encodes the lambda repressor, thus lambda repressor
controls its own synthesis)
• If the lambda repressor wins the race with the
Cro protein, lysogeny is established and the
lambda genome is integrated into the host
chromosome.
• Integration is catalyzed by the enzyme
integrase, the product of the int gene, and
takes place in the host chromosome at
attachment site (att site)
Cro protein
• 66 aa long and forms a dimer that binds the operator sites OR and
OL, blocking transcription from the PR and PL promoters.
• If Cro protein wins the race with lambda repressor, it blocks
synthesis of lambda repressor and prevents integration of the
lambda genome into the host chromosome.
• By the time synthesis of lambda repressor is blocked, another
regulatory protein called Q protein has accumulated.
• Q promotes transcription from a promoter called PR′, and in the
presence of Q protein, the genes encoding viral structural proteins,
as well other proteins needed for virus assembly and host lysis, are
transcribed.
• Ultimately, the host is lysed and the new virions are released.
Immunity to super infection
• In a lambda lysogenic bacterium, the only viral
protein synthesized is lambda repressor.
• Thus if a new lambda phage infects the cell,
lambda repressor can bind the regulatory sites
of the incoming viral genome immediately,
and expression of all genes (and
superinfection) is blocked.
Induction
• Occurs in response to environmental factors such as UV light or
chemical mutagens that damage DNA.
• This damage alters the activity of the RecA protein (recombination
and DNA repair)
• When activated by DNA damage, RecA interacts with lambda
repressor, causing the repressor to cleave itself.
• As more and more repressor proteins destroy themselves,
transcription of the cI gene is decreased, further lowering the
amount of lambda repressor in the cell.
• Eventually the level becomes so low that initiation of transcription
of the xis, int, and cro genes occurs.
• The xis gene encodes the protein excisionase. It binds integrase,
causing it to reverse the integration process, and the prophage is
freed from the host chromosome.
• As lambda repressor levels decline, the Cro protein levels increase.
Eventually, synthesis of lambda repressor is completely blocked and
the lytic cycle proceeds to completion
THANK YOU

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LYTIC AND LYSOGENIC CASCADE.pptx

  • 1. LYTIC AND LYSOGENIC CASCADE Medhavi Vashisth, Anu Bala, Priya Sharma, Taruna Anand, Nitin Virmani, B.C. Bera, Rajesh K. Vaid National Centre for Veterinary Type Cultures, ICAR- National Research Centre on Equines, Hisar, India
  • 2. Several types of independent genetic units in bacteria integrated Plasmid vs. episome = lytic vs. lysogenic phage free form free form integrated integrated conjugation lysis Some plasmids are able to integrate into the chromosome and are thus replicated with the chromosome. Such plasmids are called episomes.
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  • 4. • After bacteriophages have reproduced within the host cell, many of them are released when the cell is destroyed by lysis. • A phage life cycle that culminates with the host cell bursting and releasing virions is called a lytic cycle, and viruses that reproduce solely in this way are called virulent viruses. • Eg. T-even phages of E. coli (Myoviridae)
  • 5. LYTIC CYCLE One Step Growth Experiment • 1939 by Max Delbrück and Emory Ellis • The reproduction of a large phage population is synchronized so that the molecular events occurring during reproduction can be followed. • A culture of susceptible bacteria such as E. coli is mixed with bacteriophage particles, and the phages are allowed a short interval to attach to their host cells. • The number of infective phage particles released from bacteria is subsequently determined at various intervals by a plaque assay
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  • 7. • During the latent period, which immediately follows phage addition, there is no release of virions. • Rise period (burst) when the host cells rapidly lyse and release infective phages. Finally, a plateau is reached and no more viruses are liberated. • The total number of phages released can be used to calculate the burst size, the number of viruses produced per infected cell. • The latent period is called the eclipse period because the virions detectable before infection are now concealed or eclipsed.
  • 8. The major events- • Adsorption to the host cell • Viral penetration of the host cell • Synthesis of viral nucleic acids and proteins • Assembly of phage particles • Release of virions
  • 9. Adsorption and Penetration • Not random • Receptor mediated- • Cell wall lipopolysaccharides, proteins, teichoic acids, flagella, and pili.
  • 10. • Tail fiber contacts the appropriate receptor • Baseplate settles down on the surface • Binding is due to electrostatic interactions and is influenced by pH and ions such as Mg2+ and Ca2+ • Tail sheath becomes shorter and wider (24 to 12 rings) • Core is pushed through the bacterial wall • The baseplate has gp5 (lysozyme): penetration through peptidoglycan layer • Linear DNA is extruded from the head, through the tail tube, and into the host cell
  • 12. Early mRNAs – • Synthesis of protein factors and enzymes to takeover cell machinery. • Virus-specific enzymes degrade host DNA to nucleotides • Halting host gene expression DNA replication – several oris, bidirectional
  • 13. Early genes transcription – • By host RNA polymerases & σ70 • Virus mediated transfer of ADP-ribose from NAD to one α-subunit of RNA polymerase. • Host gene transcription is inhibited. • Second α-subunit receives an ADP-ribosyl group - turns off some of the early T4 genes except motA- transcription of late genes • One of the later genes encodes σgp55. This sigma factor helps RNA polymerase bind to late promoters and transcribe late genes. • Restriction – glucosylated HMC & methylation
  • 14. Late mRNAs- Directs the synthesis of three kinds of proteins: (1) phage structural proteins (2) proteins that help with phage assembly without becoming part of the virion structure (3) proteins involved in cell lysis and phage release.
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  • 16. Release of the phage particles • Two proteins are involved. • One directs the synthesis of an enzyme that attacks peptidoglycan in the host’s cell wall. It is sometimes called lysozyme. • Another protein called holin creates holes in the E. coli plasma membrane, enabling lysozyme to move from the cytoplasm to the peptidoglycan. Endolysins – • Endo-β-N-acetylglucosaminidase • N-acetylmuramidase (lysozyme) • Endopeptidase • N-acetylmuramoyl-L-alanine amidase • ϒ-D-glutaminyl-L-lysine endopeptidase
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  • 19. Temperate phages that have two reproductive options: • they can reproduce lytically as do the virulent phages, • or they can remain within the host cell without destroying it. • Lysogeny- relationship between a temperate phage and its host. • Prophage- The form of the virus that remains within its host. • Lysogens (Lysogenic bacteria)- The infected bacteria
  • 20. Two distinctive characteristics- • These cannot be reinfected by the same virus (immunity to superinfection) • Under appropriate conditions they lyse and release phage particles (Induction) Lysogenic conversion • This occurs when a temperate phage induces a change in the phenotype of its host. • Often involve alterations in surface characteristics of the host. Eg. Salmonella and epsilon phage • Give the host pathogenic properties. Eg. Corynebactium diptheriae and phage β
  • 21. Two advantages : • It allows a virus to remain viable within a dormant host. • In such situations, a prophage would survive but most virulent bacteriophages would not be replicated as they require active cellular biosynthetic machinery. • When there are many more phages in an environment than there are host cells, a situation virologists refer to as a high multiplicity of infection (MOI). • In these conditions, lysogeny allows for the survival of host cells so that the virus can continue to reproduce.
  • 22. • Illustrated by bacteriophage lambda. • Lambda is a double-stranded DNA phage that infects the K12 strain of E. coli. • Its DNA genome is a linear molecule with cohesive ends. • 40 genes. Most genes are clustered according to their function, with separate groups involved in head synthesis, tail synthesis, lysogeny, DNAreplication, 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 lysogeny. • Regulation of appropriate genes is facilitated by clustering and coordinated transcription from the same promoter.
  • 23. • Two regulatory proteins – • Lambda repressor (product of the cI gene) - lysogeny • Cro protein (product of the cro gene) – lytic cycle • If lambda repressor prevails, the production of Cro protein is inhibited and lysogeny occurs. • If the Cro protein prevails, the production of lambda repressor is inhibited and the lytic cycle occurs. • This is because the lambda repressor prevents transcription of viral genes, while Cro does just the opposite: it ensures viral gene expression.
  • 24.
  • 25. Lambda Repressor • 236 aa long and folds into a dumbbell shape with globular domains at each end. • One domain binds DNA while the other binds another lambda repressor molecule to form a dimer. • Lambda repressor binds two operator sites OL and OR, thereby blocking transcription of most viral genes. • When bound at OL, it represses transcription from the promoter PL. Likewise, when bound at OR it represses transcription in the rightward direction from PR . • However, it also activates transcription in the leftward direction from the cI promoter PRM (cI encodes the lambda repressor, thus lambda repressor controls its own synthesis)
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  • 27. • If the lambda repressor wins the race with the Cro protein, lysogeny is established and the lambda genome is integrated into the host chromosome. • Integration is catalyzed by the enzyme integrase, the product of the int gene, and takes place in the host chromosome at attachment site (att site)
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  • 30. Cro protein • 66 aa long and forms a dimer that binds the operator sites OR and OL, blocking transcription from the PR and PL promoters. • If Cro protein wins the race with lambda repressor, it blocks synthesis of lambda repressor and prevents integration of the lambda genome into the host chromosome. • By the time synthesis of lambda repressor is blocked, another regulatory protein called Q protein has accumulated. • Q promotes transcription from a promoter called PR′, and in the presence of Q protein, the genes encoding viral structural proteins, as well other proteins needed for virus assembly and host lysis, are transcribed. • Ultimately, the host is lysed and the new virions are released.
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  • 34. Immunity to super infection • In a lambda lysogenic bacterium, the only viral protein synthesized is lambda repressor. • Thus if a new lambda phage infects the cell, lambda repressor can bind the regulatory sites of the incoming viral genome immediately, and expression of all genes (and superinfection) is blocked.
  • 35. Induction • Occurs in response to environmental factors such as UV light or chemical mutagens that damage DNA. • This damage alters the activity of the RecA protein (recombination and DNA repair) • When activated by DNA damage, RecA interacts with lambda repressor, causing the repressor to cleave itself. • As more and more repressor proteins destroy themselves, transcription of the cI gene is decreased, further lowering the amount of lambda repressor in the cell. • Eventually the level becomes so low that initiation of transcription of the xis, int, and cro genes occurs. • The xis gene encodes the protein excisionase. It binds integrase, causing it to reverse the integration process, and the prophage is freed from the host chromosome. • As lambda repressor levels decline, the Cro protein levels increase. Eventually, synthesis of lambda repressor is completely blocked and the lytic cycle proceeds to completion