Bacteriophages Flashcards
Describe the replication process of PhiX174
The pilot protein H binds to a lipopolysaccharide in the bacterial cell wall and injects the ssDNA (+) genome into the cell. As soon as it enters it is coated in single stranded binding protein apart from the hairpin loop otherwise the A* protein would destroy the DNA before it could be primed properly. Host helicase and pre-primosome assemble on the hairpin loop and every 1500 nucleotides the host primase adds 10-15 RNA nucleotides. Host DNA polymerase synthesises the new strand as it does so, the SSBP falls off. RNA nucleotides are removed and gaps are filled by DNAP. Once the (-) strand has been synthesised, A protein makes a nick in the dsDNA and host DNAP starts copying the DNA as a rolling circle. As it is synthesised, the (+) strand is coated with binding protein.
How does PhiX174 leave the host cell?
Protein E inhibits peptidoglycan which lyses the host cell.
When does the lambda phage enter the lysogenic cycle?
Nutrients are low, the temperature is low and there is high multiplicity of infection.
What genome does PhiX174 have?
ssDNA (+)
What genome does the lambda phage have?
dsDNA
What genes are expressed during lysogeny by lambda phage and what induces the switch to the lytic cycle?
During lysogeny only the lambda repressor is made from the cl gene. Rec A (a protease) levels are low.
However during high stress or UV, RecA is increased which cleaves and destroys the lambda repressor.
This results in the expression of cro. Cro represses the lambda repressor and enters the lytic cycle.
Which bacteriophage genome enter linearly?
Lambda phage. The genome has cos sites which anneal with each other making it circular again.
How does the lambda phage genome enter the cell?
Binds to the lambda receptor using the lambda J protein in the tail tip. The linear phage genome then enters through the outer membrane and DNA binds to sugar transport protein ptsG.
What are the similarities and differences between lambda phages and retroviruses?
Both use their own integrases but the bacterial one is called a recombinase.
Differences:
Retrovirus integrates randomly whereas bacteriophages integrate at specific sites - the attB sites in the E.coli genome. Unlike the retrovirus, the phage must excise itself t replicate as a circular strand.
How is transcription regulated in bacteriophage T4?
Upon cell entry, early genes are transcribed first as its promoter binds most strongly to the host ribosome. The RNAP is then modified to bind to the middle and then late promoters.
What do the early proteins of bacteriophage T4 include?
Proteins to repair the hole in the bacterial cell wall. DNAse to degrade host DNA (virus contains modified DNA bases to protect itself). Replication proteins. Virus specific DNAP.
How is the life cycle of bacteriophage T4 so quick?
Replication starts at several places of the linear dsDNA for speed. These branched concatamers can then be packaged into multiple heads simultaneously by an ATP-dependent loading enzyme.
How does bacteriophage T4 enter and leave the cell?
Phage binds to lipopolysaccharides on bacterial outer membrane. The whole sheath contracts punching through the outer membrane. Lysozyme at the tip penetrates the peptidoglycan. It then punches through the inner membrane, injecting the linear DNA.
By the time the cell lyses 100-150 particles would have been made. Two proteins are involved in lysis; gpe (lysozyme) and gpt (T4 holin) which creates a hole in the inner membrane by degrading peptidoglycan.
Name as many bacteriophages as possible without the tail.
PhiX174, M13, MS2, Pseudomonas bacteriophage Phi6.
How does MS2 bacteriophage regulate gene expression?
Temporal expresssion and ribosomal frameshift.
At start of infection CP and RdRp are made. As CP builds up, they bind to the mRNA to stop the RNAP from binding to it.
Ribosomal frameshift is used to express the lysis protein as only a small amount of it is required.
Which bacteriophage genus is the only one to have both a capsid and envelope?
Cystovirus
Which bacteriophage has a segmented genome and of which nucleic acid?
Pseudomonas phi 6. dsRNA.
How does pseudomonas phi 6 enter the cell?
The P3 protein on virus binds to the pili which then contracts, bringing into closer contact with the host cell outer membrane.
P6 mediates fusion of both of the membranes.
Endopeptidase digests the peptidoglycan. Core enters the bacterial cell to keep the dsRNA hidden from host antiviral mechanisms.
How does pseudomonas phi 6 replicate?
The + strands are pushed out of the core into the cytoplasm. As it is translocated, replication takes place inside the virion by the RNAP.
Cellular machinary is used to translate the l+ strand which encodes P1, P2, P4 and P7 - these proteins form a polymerase complex which packages one L+, M+ and S+ into new capsid. Once inside the capsid they are converted into double strand by RdRp (P2).
What are two examples of arms races that have taken place between bacteriophages and bacteria?
1) Bacterial restriction endonucleases cleave any unmethylated DNA. Bacterial chromosome is methylated by methylase in a plasmid.
Bacteriophage T4 ligase enters with methylated cytosine.
E.coli has a modified R/M system to recognise phage DNA.
Phage has glucosylated genome which overcomes the new system.
GmrS-GmrD (glucose modified) restriction endonuclease by the bacteria also formed to restrict glucosylated bacteriophage.
Bacteriophage synthesises IPI protein which disables the GmrS-GmrD system.
The GmrS-GmrD fusion is a defence against IPI protein.
Bacteria infect by an unknown mechanism.
2) CRISPR/Cas
When a phage infects bacteria, somehow a section of it may become incorporated into the CRISPR locus of the bacterial genome. This locus is made up of unique spacers (from previous infections) and repeat regions. Upstream of it also encodes the Cas9 enzyme.
CRISPR locus is synthesised as a long mRNA (1-12 spacers) which is then cleaved into smaller segments. If invading DNA is complementary to a segment it is cleaved by Cas9. Bacterial DNA is protected by the repeat region which also has a 3’ hairpin loop.
Bacteriophages can overcome this by a) positive selection on protospacer region so it is no longer complementary and b) by having regions either side of the protospacer so it also complementary to the repeat regions and protected.
Give three examples of bacteriophages causing disease in humans
Cholera - Vibrio cholerae infected by CTX phage
Botulinum - Clostridium botulinum infected by clostridial phages
Diptheria - Corynebacterium diptheriae by beta phage
Describe how cholera spreads
When infected by the CTX phage, the cholera toxin is expressed. This leaves the cell and binds to epithelial cell in gut. The A protein is endocytosed and switches on the G proteins. This results in increase cAMP. cAMP turns on the CF transporter so it is always on. CF transporter pumps out Cl- and water resulting in diarrhoea.
What happens in botulinum and why?
The botulinum toxin is endocytosed in the nerve and cleaves the SNARE proteins. SNARE proteins are needed for fusion of the neurotransmitter vesicle and the membrane to signal muscle contraction. Without this fusion get paralysis of muscle fibres.
What happens in diptheria?
The diptheria toxin binds to receptors abundantly found on the kidney. It is internalised and the endosome is acidified. This causes a conformational change in the toxin, turns off elongation factor and kills the cell.
Give four advantages of using phages instead of antibiotics to treat bacterial infections
- Bacteria does not need to be growing (antibiotics only work on dividing bacteria)
- It is self-dosing, the phage will replicate until no bacteria left and then stop
- Phages are specific to one bacteria so it won’t kill all bacteria in gut, some of which may be useful
- Phages can evolve with the bacteria
Give five disadvantages of using phages instead of antibiotics to treat bacterial infections
- Requires lots of testing so licensing is a problem
- Might evolve to become pathogenic itself
- May result in an immune response to the phage
- Cost - once one has been generated, people can culture it themselves so not much money
- Timing - people ill with a bacterial disease will not have time to test the phage
