Bacteriophage Lambda Flashcards
(40 cards)
What are the minimal functions of a bacteriophage?
- Protection of nucleic acid
- Delivery of nucleic acid
- Conversion of infected cell to produce phage
- Release of phage
Briefly describe the steps of the lytic cycle.
1) Phage attaches to bacterium, with the tail recognising receptors on the cell surface
2) DNA injected into bacterium (nucleic acid may have to be unpackaged if filamentous)
3) Enzymes for DNA synthesis are made and replication begins (early development)
4) Genomes, heads and tails are made, and the DNA is packaged into the heads and the tails attached (late development)
5) Lysis occurs; the cell is broken to release progeny phages. Some filamentous phage ‘bud off’ instead of causing cell lysis.
Briefly describe the steps of the lysogenic growth cycle.
1) Similarly to the lytic cycle, the phage attaches to the bacterium and injects its DNA
2) Phage DNA is integrated into the bacterial genome
This is a slower process than the lytic cycle but less risky.
What is a virulent phage?
Always enters the lytic cycle (can not undergo lysogeny), and so these strains have often incurred mutations as entering the lysogenic cycle can be beneficial for phage survival.
What is a temperate phage?
Has the ability to enter both the lytic and lysogenic cycles.
What is a lysogen?
A bacterial cell containing DNA with incorporated phage DNA in it.
What is a prophage?
Refers to the phage DNA within the bacterial chromosome.
What is immunity in terms of the lysogenic cycle?
Immunity refers to a lysogen now being immune to infection of phage particles from the same species (second infection wont lead to lytic cycle). This is as repressor proteins have been translated that are specific to the operon on that species of phage DNA.
What is induction?
When the lytic cycle is induced from the lysogenic. This occurs due to the cleaving of the dimeric repressor protein, or by changing the concentration of the CII protein.
Describe how conditions affect whether the lytic or lysogenic cycle is entered.
Conditions favoured by lysogenic: low nutrient levels and high MOI (multiplicity of infection) i.e. greater number of phage than bacterial cells. This is as some phage will not be able to find cells to infect and thus will be exposed to the environment/nucleases. Low nutrient levels may cause the bacterial cell to go into a dormant state so lytic cycle cannot happen as can’t take over cellular machinery.
Conditions favoured by lytic: low MOI i.e. more bacterial cells than phage particles, as phage progeny will have lots of cells to infect.
How can you distinguish if a phage is virulent or temperate experimentally?
By growing phage on a bacterial lawn. If the phage is virulent then the lawn will be clear as it made ‘holes’ in the cells. If the phage is temperate then there will be an area that is clear (due to initial lytic cycle), but when the lysogenic cycle is induced the cells will grow over this clear area to make it turbid.
Why is it important that the bacteriophage lambda DNA forms a circle when in the bacterial cell?
It is needed as the promoter for the late genes (structural components) is before the S region where the circle is joined, and the late genes are after this region. If it was not made into a circle then the head and tail of the phage would not be made (would not be expressed as promoter in wrong position). The circularisation allows the formation of the operon, but this is not needed when the phage is not in the cell.
Describe the lytic cycle cascade in bacteriophage lambda.
1) Host RNA polymerase initially controls the lytic cycle and transcribes the early genes. From the first promoter to the first termination site, a set of genes are transcribed.
2) These genes are translated to regulatory proteins, and in bacteriophage lambda this protein is an antitermination factor which allows the RNA pol to carry on past the termination site (stops RNA pol from recognising terminator sequence).
3) The RNA pol is then able to transcribe the delayed early expression genes, and again regulatory proteins are subsequently translated. Also structural genes begin to be transcribed.
4) Transcription of the late genes is then allowed, and phage components can then be built.
Groups of genes are expressed in an ordered manner.
In bacteriophage lambda, the regulatory protein produced during lytic development is the antitermination factor. What other regulatory proteins are there in different phages and how do they allow transcription of the delayed early and late genes?
- A new RNA polymerase may be produced (different from the initial host polymerase) that can recognise the 2nd promoter and transcribe this next section of the DNA sequence.
- Sigma factor may be produced, which changes the conformation of the host RNA polymerase so it is able to recognise the next phage promoter sequence.
What is pN?
The N protein, the result of the transcription of the N gene (part of the immediate early genes transcribed in lytic development). It is an anti-termination factor and so allows the delayed early genes to be transcribed, as it interacts with RNA pol and stops it from recognising the first terminator sequence. Permits transcription past N and cro (early genes).
Where is the cI gene located and what does it encode?
It lies between the left and right promoters (lambda has left and right transcription units). It encodes for the repressor, and has 2 promoters (Prm and Pre).
What is pQ?
This is an antiterminator that allows the transcription of the late genes, by interacting with the RNA polymerase and preventing it from recognising the terminator sequence.
What are nut sites?
Where the N protein binds to allow the first antitermination event.
What are Prm and Pre?
They are both promoters for cI (the gene that encodes the repressor).
Prm = repressor maintenance
Pre = repressor establishment
Prm is a weak promoter and only works in the presence of the repressor protein, and thus Pre is needed initially when this is not present.
Prm is autoregulatory.
Why is the repressor needed and how does it work?
The repressor is needed to inhibit the lytic cycle when there are low nutrient levels and a high MOI. It acts on the operators Or and Ol to block the transcription of the immediate early genes (effectively turns off Pl and Pr), which inhibits the whole lytic cycle as it is a cascade and the antitermination factor required will not be produced.
How does the repressor protein allow immunity?
Free repressor dimer will bind to any newly inserted DNA in the cell if the same species of phage infects again, as it will recognise the operator sequences Or and O, and this prevents the lytic cycle from occurring. If a different species infects, the repressor present will not recognise the operator sequences.
What are the 2 distinct domains in the repressor and what happens if the dimers are cleaved?
N-terminal domain is involved in binding and C-terminal domain involved in dimerisation. The dimer allows it to effectively bind to the operator, and when it is cleaved (e.g. by nucleases or UV light), there is a reduction in binding affinity and some of the operator sequence is exposed, allowing RNA to still bind to the promoter and thus allowing transcription and inducing the lytic cycle.
How does the CII protein make Pre a stronger promoter?
Pre is a poor match to the consensus sequence and so is not recognised by most RNA polymerases. CII binds to the host polymerase and changes its conformation, allowing RNA pol to recognise the Pre. Therefore, the cI gene encoding the repressor is expressed and the lytic cycle inhibited.
What is HFLA and why does its presence mean that the lytic cycle is likely to be induced?
HFLA is a bacterial protease that can degrade CII, meaning that it cannot bind to RNA pol. This means that the expression of cI is reduced (as Pre continues to be a weak promoter), so less repressor is translated and the lytic cycle will probably be induced.
