Phage replication strategies Flashcards
what is bacteriophage
virus that infects bacteria and replicates inside it
why is bacteriophage studied
increase our understanding of fundamental biology
what do bacteriophages provide
a way to transfer genes between bacteria: useful in the laboratory and nature
what are bacteriophages exploited for uses
- tools in molecular biology
- biocontrol agents in agriculture e.g. to control infections
- vehicles for vaccine delivery
- detection and killing of pathogenic bacteria (using phages instead of antibiotics)
how are bacteriophages transferred
Can transfer between bacteria through conjugation
Can use phages to transfer bacteria
what are the bacteriophages that infect E.coli
ΦX174
M13 phage
Phage Lambda
Phage T4
what is ΦX174 (Phi X174)
Small and simple structure with no tail
what DNA does ΦX174 (Phi X174) have
Circular ssDNA
what is the structure of ΦX174 (Phi X174)
Head region
DNA inside of it
what helps ΦX174 (Phi X174) mature
proteins help it mature
what is a procapsid
immature phage
what happens when ΦX174 (Phi X174) mature
lose proteins
what is the structure os M13
Filamentous structure
Surrounded by coat proteins
what DNA does M13 phage have
circular ssDNA
what is phage lambda structure
Complex structure with a head and tail
long (injected into bacterium)
what is phage Lambda DNA
linear dsDNA
what is phage T4 structure
Complex structure with a head and tail
bigger fibres
has a tail
what is the structure of phage T4 DNA
linear dsDNA
what is bacteriophage lytic life cycle
Attach to bacteria surface, inject their DNA inside it (only part of the phage e.g. the tail inserts into the host)
Makes lots of copies of itself inside, replicate - copies made inside, cell eventually bursts and the phages are released from inside and these can go on to infect
what do bacteriophages contain
either RNA or DNA enclosed in a protein coat
how do bacteriophage infect bacteria
by attaching to a receptor on their surface and transmit their nucleic acid inside
what genes do bacteriophages have
have early, middle and late genes
what happens to the bacterium when inside the bacterial host cell
Once inside the bacterium the nucleic acid is replicated and new phage particles are assembled
what happens when host cell lyse
many new phage particles are released
do all phages enter bacteriophage lytic life cycle
some phages (such as lambda) may not always enter the lytic cycle and may instead establish lysogeny (stable relationship with the host cell), and M13 phage exits without lysis
what are the bacteriophages sets of genes for
allow their replication and subsequent assembly of new phage particles inside a bacterium
what are the bacteriophage replication of ssDNA
ΦX174 and M13
what are the bacteriophage replication of dsDNA
phage Lambda
phage T4
what does theta form replication require
circular DNA template RNA primer BUT IN BACTERIA DONT NEED RNA PRIMER
RecA
what is theta form replication like
Can be uni- or bi-directional
what does theta form replication make
2 circular DNA molecules
what happens in rolling circle replication
- concatamers represent linearly joined copies of the plasmid genes
- homologous sequences align and RecA promotes recombination and releases free plasmid molecules
in bacteria is rolling circle replication uni- or bi-directional
In bacteria some plasmids can use a different method of replication that is NOT bidirectional
how does DNA synthesis of rolling circle replication occur
occurs from ‘3 OH of DNA molecule
what is needed to finish replication
RecA protein
what replication is used for conjugation and bacteriophage
rolling circle replication
how is phage replication prepared
entering a bacterium phages need to prepare DNA or RNA for replication – it needs to be converted to the replicative form (RF)
ssDNA must become dsDNA
Linear DNA must become circular
how is ΦX174 and M13 replicated
infecting ss viral genome
theta form replciation copies RF dsDNA
replication (bi-directional ori)
gene II nicks +strand
rolling circle replication makes new ssDNA +strand genomes
gene II nicks completed and +strand circularisation of completed strand
what is ΦX174 and M13 replication similar to
RCR during plasmid conjugative transfer
what does ΦX174 and M13 genomes contain
small and circular ssDNA, and contain overlapping genes
what does the viral particle of ΦX174 and M13 contain
+strand
what happens after ΦX174 and M13 infection
host cell proteins perform complementary strand synthesis to make dsDNA (RF)
what forms the RF copies in ΦX174 and M13 replication
Theta form replication is then used to make copies of RF
what are the RF copies from ΦX174 and M13 used to make
more + strands by rolling circle replication (RCR)
what happens to new +strands in ΦX174 replication
packaged into capsids (ΦX174) to make new phage particles
what happens to new +strands in M13 replication
assembled with coat proteins at the membrane (M13) to make new phage particles
what are M13 new ssDNA +strands stabilised by
binding proteins inside the host cell and transferred to the cell membrane
what is the end of the M13 phage particle assembly
new viral DNA is extruded from cell and coat proteins are added during extrusion, finally releasing a complete new M13 phage particle
what must first happen to Lambda and T4 phages before replication
Linear dsDNA must first become circular
how is DNA packaged to new phage
DNA for packaging into new phage heads is synthesised by rolling circle replication (concatamers method)
how are phage capsid forms
Phage capsids are pre-formed ready to package the DNA
how is the complete phage for Lambda and T4 phage complete
phage tails and fibers are then connected to complete the new phage particle
how is Lambda DNA circularised
Homologous 12 bp single stranded sticky ends to linear genome - Cohesive ends (cos)
entering cell sticky ends anneal to form circular dsDNA = Replicative Form (RF)
what happens in the Lambda replication (lytic cycle)
- Linear dsDNA circularised by ligating cos sticky ends
- Theta form replication makes RF copies
- Rolling circle replication produces linear concatamers of dsDNA genome with identical ends (cos)
how is Lambda phage packaged
Linear DNA packaged into empty capsid head until cos site enters
Enzyme localised to head structure recognises cos site and cuts DNA asymmetrically – leaving 12bp overhangs
amount of DNA packaged into head is whatever is between cos sites
what is Int
integrase
what are the two stages of lysogeny
integration
excision
what happens in lysogeny integration stage
Integrase catalyses site-specific recombination between attP and attB 15 bp core sequences to integrate phage lambda into the bacterial chromosome
attP and attB are NOT identical, 2 different sequences are created
what are the 2 different sequences that are created by integration
attL and attR =
what is prophage
Integrated phage lambda
what happens in excision
Int needs Xis to recognise attL and attR and recombine them again
what does excision allow
induction into lytic cycle
what happens if attB is deleted from bacterial chromosome
other sequences with similarity to attB may be used at lower frequencies
alternative sites for phage Lambda attachment to bacterial chromosome
how does Lambda phage DNA effect cells in lysogeny
Phage DNA is maintained and replicated in the host cell without causing damage to the cell
how does lambda phage integration occur in lysogeny
via semi-homologous recombination
what is phage attachment in Lambda lysogeny
Phage attachment site is partially homologous to the chromosomal integration site (attP = phage sequence; attB = bacterial chromosome sequence)
what does integrase do
catalyses the integration recombination
what does integrase need
needs Xis to catalyse the excision recombination and allow induction of lytic cycle
can lysogens form from Lambda mutants
can’t integrate may still form lysogens – maintained as extrachromosomal circular DNA – can be unstable though
what is the lytic/lysogeny switch
Decision to follow either the lytic or the lysogenic pathway is made early after infection (involves early genes)
Determined by the balance between cI and cro proteins
if cro more than cI what happens
lytic cycle
if cI more than cro what happens
lysogenic cycle
what does the cI repressor do
expressed early from PM
switches off PL and PR leading to down regulation of Cro
Promotes LYSOGENIC cycle
what does the cro (anti-cl repressor) do
expressed early from PR
antagonises cI due to much weaker repression of PL and PR
Promotes LYTIC cycle
what are the additional genes that are activated after pathway chosen
delayed early genes
what happens if lysogeny is followed
cII and cIII become expressed, which increase cI repressor expression and help establish and stabilise the lysogenic state
what mechanism are lysogeny and lytic cycle
Dominance of either cro or cI will further increase their expression: positive feedback
what does DNA damage cause
induction of phage Lambda
what happens when DNA is damaged
accumulation of activated RecA (SOS repair)
cI is broken down
Cro accumulates
DNA replication activated for the lytic cycle
what are the T4 phage ends like
has homologous ends
what happens in homologous recombination
homologous recombination between them catalysed by RecA allows circularisation to replicative form
what happens in phage T4 replication
- RecA-mediated homologous recombination makes circular RF
- Theta form replication makes RF copies
- rolling circle replication makes concatamers pf phage genome for packaging new phage particles
what happens in phage T4 terminal redundancy
- RCR makes linear concatemers of dsDNA genomes
- genome packaged into empty capsid heads until they become full - more than one whole genome so ends are duplicated
- concatamers cut and next portion of DNA packaged into another phage head
what does RCR do in phage T4
makes linear concatamers of ds DNA genome
what is the terminal redundancy
170kbp linear packaged genome with 2 ends having identical sequences of 1.6kbp
what is circularly permuted
in linear form each phage genome may start and end with different genes
what are the common features to all 4 phages
dsDNA circular
need to be in a host cell to replicate
theta form - initial copies of their genome
RCR
what does cro protein do
action promotes phage Lambda lytic cycle
what does int protein do
catalyse recombination of attB and attP
what does cI (c1) protein do
action promotes phage Lambda lysogenic cycle
what does Xia protein do
assists Int to catalyse recombination of attL and attR
what does RecA protein do
accumulates upon phage Lambda induction in response to DNA damage, promotes DNA recombination
what is cos
unpaired gene sequence found at both ends of Lambda phage genome
what are the bacteriophage applications
phage display
transduction
phage-based therapies
what are the useful applications in phage display - M13
Protein-protein interactions
Drug discovery
Monoclonal antibody discovery
what happens to the phage genes to be packed into M13 cloning vectors
Some phage genes can be deleted and other genes can be cloned in, which will be packaged into phage particles
what happens to the cloned sequences - M13 cloning vectors
cloned sequences are fused to capsid protein resulting in their display on the surface of the phage particle
what is biopanning used for
identify phage peptides with desired binding properties
what is the phage display selection cycle
phage displaying antibody
selective binding using an immobilised antigen
wash off unbound phage
elute bound phage
amplification of phage in E. coli
next screening round - repeat for enrichment
what are the two types of transduction
generalised
specialised
what can phages mediate between bacterial cells
horizontal gene transfer between bacterial cells
what is generalised transduction
Segment of DNA is carried from one bacterial cell to another by a bacteriophage
Bacteriophage attaches to bacterial host cell and injects its nucleic acid
Phage enzyme made that breaks down host DNA to smaller fragments
Phage DNA is replicated, phage coat proteins are made
During formation of the mature phage particles, a few phage heads may surround fragments of bacterial DNA instead of phage DNA
Phage particle carrying bacterial DNA infects another cell, transferring bacterial DNA to the new cell, when bacterial DNA introduced into new cell, can become integrated into the bacterial chromosome, so transferring genes to recipient
Cell then multiplies
what is specialised transduction
transfer of a few species genes using a phage
Phage injects DNA into bacteria host cell
This integrates into a specific site in the host chromosome
When an E.coli culture carrying the Lambder phage is induced, phage particles are produced
On rare occasions part of the bacterial DNA is taken that is attached to the phage DNA and some phage DNA is left behind. The phage are ineffective as they do not carry the whole phage genome, but can still infect other cells
The defective phage can attach to another bacterial cell and the DNA can be injected and integrated into the new host chromosome
Only bacterial genes located near the integration site can be transduced
what is phage therapy an alternative to
antibiotics
