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Virology > 2 Bacteriophage > Flashcards

2 Bacteriophage Flashcards

1
Q

Classification of phage

A
  • Variation in phage structure

- Variation in receptors

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2
Q

lysogenic phage example

A

lambda

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3
Q

lytic phage example

A

T4

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4
Q

Bacteriophage (phage)

A

viruses that specifically infect bacteria

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5
Q

where are bacteriophage

A

common in all natural environments

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6
Q

what does phage presence relate to

A

to numbers and types of bacteria present

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7
Q

what does diversity indicate

A

Diversity detected due to lack of eukaryotic competitors that will eat bacteriophage as a food source

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8
Q

how many groups of bacteriophage are there

A

at least 12 distinct groups

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9
Q

how are bacteriophage classified

A

on basis of morphology & nucleic acid type

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10
Q

what morphology is only found in bacteriophage

A

tailed viral morphology

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11
Q

tailed phage examples

A

Siphoviridae (lambda)

Myoviridae (T4)

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12
Q

no envelope phage examples

A

Siphoviridae (lambda)
Myoviridae (T4)
Inoviridae (M13, Fd)

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13
Q

examples of linear ds DNA

A

Siphoviridae (lambda)

Myoviridae (T4)

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14
Q

examples of circular ss DNA

A

Inoviridae (M13, Fd)

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15
Q

what are the Siphoviridae (lambda) tails like

A

Tailed phage but NO tail fibres

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16
Q

what are the Myoviridae (T4) tail fibres like

A

tailed fibres normally wrapped against tail (hinge to allow legs to fold up and stick out at the bottom of the capsid)

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17
Q

Inoviridae (M13, Fd) shape

A

flexible rod shape with variable length

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18
Q

what do phage need to be able to replicate

A
  • 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
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19
Q

factors affecting efficiency of infection

A
  • 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
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20
Q

Phage adsorption occurs in 2 steps

A
  1. Reversible interaction

2. Irreversible interaction

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21
Q

Reversible interaction phage adsorption

A
  • loose association with host cell surface

- can be stabilised by the presence of metal ions

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22
Q

what are the metal ions that stabilise reversible interaction phage adsorption

A

Ca 2+
Mg 2+
Zn 4+

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23
Q

irreversible interaction phage adsorption

A
  • virus binds to receptors
  • Binding between phage and host cell receptor protein
  • often associated with conformational changes in phage structure
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24
Q

Host cell receptors

A

Most phage receptors serve essential or beneficial functions & therefore are not easily lost from the population

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25
Q

phage location of receptors

A

Other phage have developed polysaccharide depolymerases to break down capsular layers, sinks to bacterial cell surface and finds receptors

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26
Q

gram negative bacteria location of receptors

A

LamB

LPS

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27
Q

prokaryotic cell surface location of receptors

A

pili

flagellum

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28
Q

what is phage infection is influenced by growth conditions

A

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

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29
Q

how is the insertion of nucleic acid different in gram positive

A

Peptidoglycan (PG) forms rigid barrier to penetration

In Gram-positive hosts, PG is too thick to penetrate directly (need enzymes)

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30
Q

what do phage use for insertion of nucleic acid into cell

A

Phage use PG degrading enzymes (lysozymes or lysins) to make small holes in PG
- e.g. Phage T4 lysozyme

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31
Q

what do enzymes do in phage

A

Enzymes drill holes through PG layer

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32
Q

what does the tail do in nucleic acid insertion

A

Tail structures often facilitate penetration (tail contracts and penetrates the cell)
- Those with contractile tails have a “syringe” type of mechanism

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33
Q

example of phage for nucleic acid insertion

A

Phage T4 (Myovirus)

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34
Q

Phage T4 infection process

A
  1. Binds to LPS via tail fibres, locates receptor (OmpC) by surface translocation
  2. Once found OmpC will irreversibly bound, tail contracts and tail core enzymes penetrates the PG
  3. Contraction drives tail tube into cell
  4. Nucleic acid is delivered straight into cytoplasm passing through both membranes
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35
Q

phage T4 surface translocation

A

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

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36
Q

Phage Lambda infection process - Siphovirus

A
  • 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
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37
Q

when are changes evident in Siphoviridae Tail Structure

A

Changes only evident at the molecular level

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38
Q

Bacillus phage PBS1 myovirus infection process

A
  • 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
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39
Q

what does MS2 infect

A

cells that carry a conjugative plasmid

40
Q

how does MS2 infect E.coli

A

Infects E. coli by attaching to PILIN proteinthat makes up the conjugative pilus
Pilus retracted into cell to initiate either conjugation OR infection

41
Q

where does MS2 phage attach

A

Attaches via a single attachment (A) protein on the phage surface

42
Q

in MS2 what is protein A attached to

A

A protein is covalently linked to the 5’-end of the phage genome (RNA) that is inside the capsid

43
Q

what part of the host cell does MS2 phage attach to

A

host cell pilus

44
Q

what happens when the pilus retracts in MS2 phage

A

A protein and RNA are pulled with it into cell, rips nucleic acids through into cell
Leaves the empty capsid outside

45
Q

what are the stages in one step growth curve

A

eclipse

latent

46
Q

eclipse stage

A

period when phage replication is occurring inside the cell

47
Q

latent stage

A

virus in cell, period before any new phage particles appear in the cell

48
Q

what happens at the start of the step growth curve

A

Titre of viruses will drop at the start when first added

49
Q

Multiplicity of infection

A

Ratio of Phage to Host cells = m.o.i

50
Q

when dies eclipse phase start

A

begins once phage nucleic acid is injected into cell

- empty capsids often seen remaining outside cell

51
Q

what is the length of Eclipse

A

time taken to synthesise & assemble new phage particles

52
Q

what happens to the optical density at the end of the growth phase

A

OD drops

53
Q

how can you measure growth - one step growth curve

A

Monitoring lysis of culture using optical density (won’t pick up very small changes)
Need to measure the number viral particles released

54
Q

what happens once phage are assembled

A

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
55
Q

when are PG enzymes made

A

enzymes (lysins) synthesised in large amounts after phage assembled

56
Q

what do lysins do

A

degrade PG by passing through these holes

57
Q

what is burst size

A

Number of phage particles produced per cell

58
Q

what is the normal range of burst size

A

normally in range of 100-1000

59
Q

what is faster phage replication or bacterial growth

A

phage

60
Q

what happens in lytic phage

A

Infected cultures will be completely lysed

61
Q

what happens when the M.O.I exceeds 5-10 (phage per cell)

A

Host cell may be killed due to action of the PG degrading enzymes associated with tail during infection

62
Q

what leads to a drop in phage titre

A
  • 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
63
Q

what does 1 plaque =

A

1 phage

64
Q

how can phage be visualised

A

Host cells and phage are mixed together in soft molten agar
Phage infects 1 cell and replicates
Results in a zone of clearing

65
Q

what does plaque morphology show

A

can give an indication of phage biology

66
Q

Turbid plaques

A

lysogenic phage

67
Q

what does plaque size relate to

A
  • growth rate of host strain
  • efficiency of phage host-receptor interaction
  • burst size
  • percentage of agar!
68
Q

what does phage DNA contain

A

unique base 5-HMC

5-hydroxymethylcytosine

69
Q

what is the effect of 5-HMC

A

Overcomes host cell restriction barrier

DNA cant be degraded

70
Q

Restriction: Host defence

A

Restriction of foreign DNA used as a defence
Bacterial cells encode restriction enzymes
Incoming Unmethylated dsDNA will be degraded by host cell restriction enzyme

71
Q

Bacterial cells encode restriction enzymes -restricting

A
  • Cut their own chromosomal DNA if it is not protected

- Cells encode a matching (cognate) methylase gene to modify and protect own DNA

72
Q

Incoming Unmethylated dsDNA will be degraded by host cell restriction enzyme effect

A
  • phage being unable to infect the cell
73
Q

Phage T4 life cycle (1)

A 
Degradation of host cell unmodified DNA
Lysozyme inhibited 
cell opens
Phage can “sense” other viruses 
release phage when right time
74
Q

what gene in T4 cycle must not be made too early

A

lysin

75
Q

when is lysozyme inhibited

A

when many phage attached to cell

76
Q

when is the cell opened in T4 cycle

A

Only open cell when phage particle fully produced

77
Q

when are phage released in T4 life cycle

A

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

78
Q

pathways of phage replication

A

lysogenic

lytic

79
Q

IE genes

A

determine decision between lytic/lysogenic pathways

80
Q

cI gene

A

establishes lysogeny

81
Q

DE genes

A

early DNA replication events

82
Q

LYSOGENIC cycle

A

cI repressor:

  • expressed early from PM
  • switches off PL and PR leading to downregulation of Cro
  • Promotes the LYSOGENIC cycle
83
Q

LYTIC cycle

A

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
84
Q

Bacteriophage λ life cycle: Lysogeny

A

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

85
Q

how do ends join in lysogeny cycle

A

Ends of λ genome join by complementary base pairing of “cohesive” ends

86
Q

phage-encoded genes in bacteriophage - lysogeny life cycle

A

CI protein activates the int (integrase) gene

87
Q

where is attB located

A

between two genes in E. coli chromosome

88
Q

what is gal

A

galactose utilisation

89
Q

what is bio

A

biotin biosynthesis

90
Q

what is unusual in phage attachment

A

λ attachment site is unusual

91
Q

how do many phage integrate

A

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
92
Q

when is Lysogenic phage expressed

A

Some lysogenic phage carry non-essential genes which are expressed during dormancy

93
Q

is lysogen presence beneficial

A

sometimes

94
Q

what is induction linked to

A

cell stress signals

95
Q

what is Bacteriophage λ life cycle: induction

A

site-specific recombination event is reversed and releases circular λ genomes
Phage replication then follows the lytic pathway

Virology (15 decks)

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