2 Bacteriophage Flashcards
Classification of phage
- Variation in phage structure
- Variation in receptors
lysogenic phage example
lambda
lytic phage example
T4
Bacteriophage (phage)
viruses that specifically infect bacteria
where are bacteriophage
common in all natural environments
what does phage presence relate to
to numbers and types of bacteria present
what does diversity indicate
Diversity detected due to lack of eukaryotic competitors that will eat bacteriophage as a food source
how many groups of bacteriophage are there
at least 12 distinct groups
how are bacteriophage classified
on basis of morphology & nucleic acid type
what morphology is only found in bacteriophage
tailed viral morphology
tailed phage examples
Siphoviridae (lambda)
Myoviridae (T4)
no envelope phage examples
Siphoviridae (lambda)
Myoviridae (T4)
Inoviridae (M13, Fd)
examples of linear ds DNA
Siphoviridae (lambda)
Myoviridae (T4)
examples of circular ss DNA
Inoviridae (M13, Fd)
what are the Siphoviridae (lambda) tails like
Tailed phage but NO tail fibres
what are the Myoviridae (T4) tail fibres like
tailed fibres normally wrapped against tail (hinge to allow legs to fold up and stick out at the bottom of the capsid)
Inoviridae (M13, Fd) shape
flexible rod shape with variable length
what do phage need to be able to replicate
- protect nucleic acid from degradation
- deliver the nucleic acid inside the bacterial cell
- convert the bacterial cell to phage replication (need to take over the cell)
- allow the progeny phage to escape from the cell
factors affecting efficiency of infection
- surface antigen changes affect binding (LPS/teichoic acid)
- no binding to mutants lacking receptor
- uses cellular enzymes for replication
- RM system can destroy incoming phage
Phage adsorption occurs in 2 steps
- Reversible interaction
2. Irreversible interaction
Reversible interaction phage adsorption
- loose association with host cell surface
- can be stabilised by the presence of metal ions
what are the metal ions that stabilise reversible interaction phage adsorption
Ca 2+
Mg 2+
Zn 4+
irreversible interaction phage adsorption
- virus binds to receptors
- Binding between phage and host cell receptor protein
- often associated with conformational changes in phage structure
Host cell receptors
Most phage receptors serve essential or beneficial functions & therefore are not easily lost from the population
phage location of receptors
Other phage have developed polysaccharide depolymerases to break down capsular layers, sinks to bacterial cell surface and finds receptors
gram negative bacteria location of receptors
LamB
LPS
prokaryotic cell surface location of receptors
pili
flagellum
what is phage infection is influenced by growth conditions
Infection only occurs if host cell expresses correct surface receptor
Often maximal expression occurs during active growth - a good time to infect!
Host range may be altered by expressing new surface receptors
how is the insertion of nucleic acid different in gram positive
Peptidoglycan (PG) forms rigid barrier to penetration
In Gram-positive hosts, PG is too thick to penetrate directly (need enzymes)
what do phage use for insertion of nucleic acid into cell
Phage use PG degrading enzymes (lysozymes or lysins) to make small holes in PG
- e.g. Phage T4 lysozyme
what do enzymes do in phage
Enzymes drill holes through PG layer
what does the tail do in nucleic acid insertion
Tail structures often facilitate penetration (tail contracts and penetrates the cell)
- Those with contractile tails have a “syringe” type of mechanism
example of phage for nucleic acid insertion
Phage T4 (Myovirus)
Phage T4 infection process
- Binds to LPS via tail fibres, locates receptor (OmpC) by surface translocation
- Once found OmpC will irreversibly bound, tail contracts and tail core enzymes penetrates the PG
- Contraction drives tail tube into cell
- Nucleic acid is delivered straight into cytoplasm passing through both membranes
phage T4 surface translocation
one leg goes down onto the surface and all the others are folded up against the body, then another leg will randomly unbind, triggering the other leg to fold up, they can translocate across cell using this
Phage Lambda infection process - Siphovirus
- tailed phage with an isometric head
- Attaches to the LamB maltose receptor on the surface of the E. coli cell.
- non-contractile tail, but DNA is still injected into cell
- empty capsid remains outside cell
when are changes evident in Siphoviridae Tail Structure
Changes only evident at the molecular level
Bacillus phage PBS1 myovirus infection process
- Tail fibres are helical - Attaches to the sides of the flagellae
- Helical tail fibres wrap around thin flexible flagellum structure
- DNA is delivered into cell via hollow core of flagellum
what does MS2 infect
cells that carry a conjugative plasmid
how does MS2 infect E.coli
Infects E. coli by attaching to PILIN proteinthat makes up the conjugative pilus
Pilus retracted into cell to initiate either conjugation OR infection
where does MS2 phage attach
Attaches via a single attachment (A) protein on the phage surface
in MS2 what is protein A attached to
A protein is covalently linked to the 5’-end of the phage genome (RNA) that is inside the capsid
what part of the host cell does MS2 phage attach to
host cell pilus
what happens when the pilus retracts in MS2 phage
A protein and RNA are pulled with it into cell, rips nucleic acids through into cell
Leaves the empty capsid outside
what are the stages in one step growth curve
eclipse
latent
eclipse stage
period when phage replication is occurring inside the cell
latent stage
virus in cell, period before any new phage particles appear in the cell
what happens at the start of the step growth curve
Titre of viruses will drop at the start when first added
Multiplicity of infection
Ratio of Phage to Host cells = m.o.i
when dies eclipse phase start
begins once phage nucleic acid is injected into cell
- empty capsids often seen remaining outside cell
what is the length of Eclipse
time taken to synthesise & assemble new phage particles
what happens to the optical density at the end of the growth phase
OD drops
how can you measure growth - one step growth curve
Monitoring lysis of culture using optical density (won’t pick up very small changes)
Need to measure the number viral particles released
what happens once phage are assembled
Once mature phage particles are assembled, cell wall must be degraded to allow release
- or host cell lysis induced*
- some phage simply activate a cellular lysin
when are PG enzymes made
enzymes (lysins) synthesised in large amounts after phage assembled
what do lysins do
degrade PG by passing through these holes
what is burst size
Number of phage particles produced per cell
what is the normal range of burst size
normally in range of 100-1000
what is faster phage replication or bacterial growth
phage
what happens in lytic phage
Infected cultures will be completely lysed
what happens when the M.O.I exceeds 5-10 (phage per cell)
Host cell may be killed due to action of the PG degrading enzymes associated with tail during infection
what leads to a drop in phage titre
- Weakened cell lyses before new phage are formed
- Phage enter eclipse phase
- No new phage particles made
- Results in an overall drop in phage titre
what does 1 plaque =
1 phage
how can phage be visualised
Host cells and phage are mixed together in soft molten agar
Phage infects 1 cell and replicates
Results in a zone of clearing
what does plaque morphology show
can give an indication of phage biology
Turbid plaques
lysogenic phage
what does plaque size relate to
- growth rate of host strain
- efficiency of phage host-receptor interaction
- burst size
- percentage of agar!
what does phage DNA contain
unique base 5-HMC
5-hydroxymethylcytosine
what is the effect of 5-HMC
Overcomes host cell restriction barrier
DNA cant be degraded
Restriction: Host defence
Restriction of foreign DNA used as a defence
Bacterial cells encode restriction enzymes
Incoming Unmethylated dsDNA will be degraded by host cell restriction enzyme
Bacterial cells encode restriction enzymes -restricting
- Cut their own chromosomal DNA if it is not protected
- Cells encode a matching (cognate) methylase gene to modify and protect own DNA
Incoming Unmethylated dsDNA will be degraded by host cell restriction enzyme effect
- phage being unable to infect the cell
Phage T4 life cycle (1)
Degradation of host cell unmodified DNA Lysozyme inhibited cell opens Phage can “sense” other viruses release phage when right time
what gene in T4 cycle must not be made too early
lysin
when is lysozyme inhibited
when many phage attached to cell
when is the cell opened in T4 cycle
Only open cell when phage particle fully produced
when are phage released in T4 life cycle
do not want to be released if other competitors, only released off surface when feedback loop (between virus on outside and phage on inside), when no competitors lysosome switched then phage released
pathways of phage replication
lysogenic
lytic
IE genes
determine decision between lytic/lysogenic pathways
cI gene
establishes lysogeny
DE genes
early DNA replication events
LYSOGENIC cycle
cI repressor:
- expressed early from PM
- switches off PL and PR leading to downregulation of Cro
- Promotes the LYSOGENIC cycle
LYTIC cycle
Cro (anti-cI repressor):
- is expressed early from PR
- it antagonises cI due to much weaker repression of PL and PR
- Promotes the LYTIC cycle
Bacteriophage λ life cycle: Lysogeny
Integrate into genome mediated by phage-encoded genes
> promotes site-specific recombination between the attP (phage) and attB (bacterial) sites
> phage becomes part of the bacterial genome
ends join
how do ends join in lysogeny cycle
Ends of λ genome join by complementary base pairing of “cohesive” ends
phage-encoded genes in bacteriophage - lysogeny life cycle
CI protein activates the int (integrase) gene
where is attB located
between two genes in E. coli chromosome
what is gal
galactose utilisation
what is bio
biotin biosynthesis
what is unusual in phage attachment
λ attachment site is unusual
how do many phage integrate
Many phage integrate into a copy of a tRNA gene
- part of tRNA gene is also encoded by phage
- after integration tRNA gene is still functional
when is Lysogenic phage expressed
Some lysogenic phage carry non-essential genes which are expressed during dormancy
is lysogen presence beneficial
sometimes
what is induction linked to
cell stress signals
what is Bacteriophage λ life cycle: induction
site-specific recombination event is reversed and releases circular λ genomes
Phage replication then follows the lytic pathway
