Bacteriophage lambda Flashcards
What is the difference between bacteriophage and bacteriophages?
Bacteriophage = singular/plural of same species Bacteriophages = two or more different species
What is an alternative term for ‘bacteriophage’?
phage
What are the main functions of a bacteriophage?
- protection of nucleic acid
- delivery of nucleic acid
- conversion of infected cell to produce phage
- release of phage
Why does a bacteriophage have such a small genome?
It has minimal functions
What is the relationship between a bacteriophage and host?
A bacteriophage subverts the host function in order to replicate.
A bacteriophage cannot survive external to the host.
How are phages classified?
by structure
What is the requirement of the phage structure?
The nucleic acid must be packaged within a protein coat.
What are the three main phage structures?
- icosahedral tailless
- icosahedral with tail
- filamentous
What is an icosahedron?
a quasispherical polyhedron, having twenty triangular faces
Describe an icosahedral tailless phage
The highly compact nucleic acid is located within the capsid, which is in the shape of an icosahedron.
Describe an icosahedral phage with tail
The icosahedral head containing the compact DNA has a tail attached to it.
Describe a filamentous phage
The nucleic acid, which has an extended helical form, is embedded in a cylindrically shaped capsid
What structure is bacteriophage T4 an example of?
icosahedral phage with tail
Why is the icosahedral phage evolutionarily limited?
due to the fixed structural requirements of the head (and tail, if there is one)
define ‘virulent’
always goes through lytic life cycle
define ‘temperate’
- goes through either the lytic or lysogenic cycles
- most bacteria are temperate
define ‘lysogen’
bacterial genome contains a latent form of the phage genome (prophage)
define ‘prophage’
incorporated into the bacterial genome
define ‘immunity’
no further infection possible
define ‘induction’
phage genome is excised from the bacterial genome and the lytic cycle begins
When is lysogeny favoured over lysis?
- low nutrient availability
- high MOI
What does MOI stand for and what is it?
- multiplicity of infection
- the average number of virus particles infecting each cell
Why is lysogeny favoured under low levels of nutrients?
- bacterium is in dormant state
- mRNA degraded and bacterium metabolises at a low level
- therefore lysogeny occurs, since there is no way for the bacterium to induce the lytic cycle
What is a plaque?
clear zones formed in a lawn of cells due to lysis by phage
How does plaque appearance indicate whether lysis or lysogeny is taking place?
- at a low MOI, a cell is infected with a single phage and lysed, releasing progeny phage that can then infect neighbouring cells, lysing them
- this results in a circular area of cell lysis in a turbid lawn of cells
- when a temperate phage infects a population of cells, each phage produces a plaque with a ‘bull’s eye’ morphology, comprising of a turbid centre surrounded by a ring of clearing
- lytic growth is favoured when cells are growing slowly and the MOI is high
- initially, the nutrients are plentiful so the bacteria grow rapidly and the MOI is low, so the phage grow lytically
- after several cycles, the local MOI increases and most of the cells are lysed, producing a plaque in the lawn of cells
- as the lawn becomes saturated, the rate of growth slows and, since lysis requires rapid metabolism, the plaque stops increasing in size
- however, any lysogens that formed in the centre of the plaque are immune to lysis and continue to grow, since they do not have to compete with nearby cells for nutrients
What is signified by clear and turbid plaque appearance?
clear = cells have undergone lysis
turbid = bacteria are unaffected or have undergone lysogeny
cells that have undergone lysogeny do not appear as dense as the agar
Describe the bacteriophage genetic information
- linear dsDNA
- circularises after it is injected into host cell
- 48 514 bp
- 12 bp cohesive ends
How does the lambda DNA overhang compare to that produced by restriction enzymes?
It is much longer
How are genes arranged on the lamdba DNA?
Genes are clustered by function eg. for DNA replication
Why is it important that the DNA circularises?
The late genes form a single transcription unit.
Outline the main stages of lytic growth
- infection: the phage must make contact with the bacterium via interaction with cell surface proteins; DNA injected into the bacterium
- early development: enzymes for DNA synthesis begin; replication begins
- late development: genomes, heads and tails are made; assembly proteins are synthesised to assist in constructing the particle; DNA is packaged into heads and the tails are attached; nucleic acid is extruded through the cell wall and some components are added
- lysis: the cell is broken to release progeny phages
How is lytic development controlled by a cascade?
Gene expression at one stage is necessary for protein synthesis at the next.
Summarise how lytic development is controlled by a cascade
- immediate early genes are transcribed by the host RNA polymerase; these include regulators, which are required for the expression of the middle set of phage genes
- expression of delayed early genes typically starts as soon as the regulator protein coded by the early gene(s) is available
- the expression of the initial set of early genes may or may not continue, depending on the nature of the control circuit
- when the replication of phage DNA begins, the late genes are expressed; their transcription is usually arranged by embedding an additional regulator gene within the previous set of genes
What is the effect of an antitermination factor?
It causes the host RNA polymerase to ignore the first terminator that it encounters.
What is N an example of?
an antitermination factor
What are the two immediate early genes?
N and cro
To which direction are N and cro transcribed?
N is transcribed towards the left
cro is transcribed towards the right
What are P(L) and P(R)?
P(L) = transcription towards the left P(R) = transcription towards the right
What are the main stages of gene expression in the lytic cycle?
- early: host RNA polymerase transcribes N and cro from P(L) and P(R)
- delayed early: pN permit transcription from the same promoters to continue past N and cro
- late: transcription initiates at P(R) (between Q and S) and pQ permits it to continue through all the late genes
What is the main difference between protein synthesis in the lysogenic cycle compared to that in the lytic cycle?
Instead of genes being transcribed in a set order, as in the lytic cycle, DNA is integrated into the host genome in lysogeny.
What is the effect of cro?
Cro inhibits the lysogenic pathway
What is the effect of cII and cIII?
cII and cIII are positive regulators that activate the expression of cI
What is a nut site?
N utilisation site; site of N binding to prevent termination replication
How is N removed from the nut site?
N is picked up by RNA polymerase
What is P(RM) and P(RE)?
P(RM) = promoter right maintenance P(RE) = promoter right establishments
How is the lambda repressor a negative regulator for transcription?
It binds at the operators to block transcription of the early genes by blocking the binding of RNA polymerase. The immediate early genes trigger the regulatory cascade; therefore their repression prevents the lytic cycle from proceeding.
How is immunity achieved?
The repressor binds to DNA injected into the lysogen, preventing the transcription of phage DNA.
What is the effect of the repressor binding at O(L)?
The repressor prevents the expression of gene N and all leftward early genes.
What is the effect of the repressor binding at O(R)?
The repressor prevents the expression of cro and other rightward early genes
What is the structure of the lambda repressor?
The repressor is comprised of a 27kDa dimer, each with two distinct domains.
What is the function of the two domains of the repressor?
- the C-teminal domains are involved in dimerisation and bind to one another
- the N-terminal domains bind DNA
Can C- and N- terminal fragments carry out their functions?
The C-terminal fragment can form oligomers.
The N-terminal fragment can bind the operators, although at a lower affinity than the intact repressor.
What are oligomers?
polymers whose molecules consist of relatively few repeating units
Why is dimerisation important?
to hold the N-terminal domains in the correct orientation
What happens to the repressor during lysogeny and during induction?
During lysogeny, monomers are in equilibrium with dimers, which bind to DNA.
During induction, cleavage of the monomers disturbs the equilibrium, so the dimers dissociate. The repressor no longer binds to DNA with high affinity and is released from the operator, causing transcription, and therefore lysis, to occur.
How does a bacterium enter into lysogeny?
- the presence of repressor protein is necessary for its own synthesis
- when lambda DNA enters a cell, RNA polymerase cannot transcribe cI because there is no repressor available to enable its binding at P(RM)
- but the absence of repressor protein means that P(R) and P(L) are available
- N and cro are transcribed, etc. and pN enables transcription to be extended
- cIII (and other genes) are transcribed on the left and cII (and other genes) are transcribed on the right
- the cII and cIII genes are positive regulators
- the cII protein acts on P(RE) to allow it to be recognised by the RNA polymerase
- P(RE) is usually a very weak promoter, but the binding of cII induces a conformational change
- P(RE) is a cI promoter
Why is the cII protein unstable in vivo?
It is the target of the proteolytic enzyme HflA (high frequency lysogenisation A)
What is the role of cIII?
to protect cII against degradation
How does transcription from P(RE) promote lysogeny?
direct effect:
cI mRNA is translated into repressor protein
indirect effect:
- transcription proceeds through the cro gene in the ‘wrong’ direction so that the resulting transcript is antisense to the cro transcript
- the complementary transcripts hybridise to inhibit the transcription of the authentic cro mRNA
- cro expression is required for entry into the lytic cycle
What is the rolling circle mechanism?
- template is circular duplex DNA
- initiation occurs on one strand
- nick at origin on one strand only
- elongation of growing strand displaces old strand
- after one revolution, displaced strand reaches unit length
- continued elongation generates displaced strand of multiple unit lengths (concatemers)
How is the icosahedral head formed?
- four different proteins make up the icosahedral head
- these make a roughly spherical shape
- a terminase enzyme attaches to the head and cuts the linear genomes from the displaced molecule
- the genome is inserted into the head
- this induces a conformational change of the head into an icosahedral shape
- the terminase makes another nick and is released from the head structure
- a small amount sticks out to interact with the tail structure
Describe the process of site-specific recombination
- nick in phage and bacteria DNA
- one end of phage DNA attaches to one end of bacterial DNA
- only one nick is made at a time
- the second nick is made
- DNA is wound around the intersome by IHF
- the integrase enzyme makes the cuts
- a different integrase ezyme makes each of the cuts, totalling four integrase enzymes
What are BOB’, POP’, BOP’ and POB’?
BOB’ = bacterial att site
POP’ = phage att site
BOP’ and POB’ = prophage att sites (based on hybrid attachment sites attL and attR)
What are attP and attB?
attP = phage attachment site attB = bacterial attachment site
What is the function of Int?
Int is required for both excision and integration of the lambda prophage
