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HELENP CBG VIROLOGY AND IMMUNITY > CBG Lecture 24: Bacteriophage > Flashcards

CBG Lecture 24: Bacteriophage Flashcards

1
Q

deine bacteriophage

A

obligate intracellular parasites that multiply inside bacteria by making use of some or all of host biosynthetic machinary

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2
Q

what do you call an obligate intracellular parasite that multiplies by making use of the host cell’s biosynthetic machinary

A

bacteriophage

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3
Q

what is the significance for using bacteriophages

A

models for animal cell viruses
gene transfer in bacteria
medical application
lysogenic conversion

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4
Q

name some uses for bacteriophages

A

models for animal cell viruses
gene transfer in bacteria
medical application
lysogenic conversion

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5
Q

what is phage therapy

A

in Eastern Europe: can order bacteriophages to treat infection: no problem with resistance and its a form of autodosing
phages can treat dysentery

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6
Q

give an example where phage therapy has been used

A

to treat dysentery

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7
Q

when phage attaches to host cell and injects DNA, what 2 pathways can happen

A

it can be lytic or lysogenic

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8
Q

what is a lytic phage aka

A

virulent

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9
Q

what is a virulent phage aka

A

lytic

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10
Q

what is a temperate phage aka

A

lysogenic

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11
Q

what is a lysogenic phage aka

A

temperate

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12
Q

what is a lytic phage

A

phage that can only kill the host cell by multiplication within it, then killing the cell by lysis

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13
Q

what is a lysogenic phage

A

can either multiply by lytic cycle or enter a dormant state where the expression of most phage genes are repressed, in a prophage

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14
Q

what is a phage that can only kill by multiplying within host cell then lysing called

A

lytic phage

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15
Q

what is a phage that can either multiply by lytic cycle or remain inactive and dormant with its genes repressed in a prophage called

A

lysogenic phage

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16
Q

what happens to the expression of most phage genes in the lysogenic phage

A

expression of most genes is repressed - -inactive

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17
Q

what is genome of a phage

A

DNA or RNA

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18
Q

what triggers conversion of lysogenic phage to lytic

A

stressor

eg. UV- host excises its virus genome making it active

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19
Q

how do phages attach to host cell

A

bind to specific receptors that are proteins or carbs in the bac cell wall

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20
Q

what is PhiX174

A

extensively studied virus = isometric with 20 traingular faces
microvirus

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21
Q

give an example of a microvirus

A

PhiX174

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22
Q

why is PhiX174 historically important

A

it was the first viral genome to be sequenced in 1977
it is plus sense ssDNA
it has a very small genome of only 5386 nucleotides
the genome is infectious by itself
its a model for fundamentals of DNA replication

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23
Q

how many nucleotides is PhiX174

A

5386 nucleotides - have a very small genome

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24
Q

what type genome does PhiX174 have

A

plus sense ssDNA - useful for studying DNA replication

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25
Q

what was the fist viral genome to be sequenced

A

PhiX174

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26
Q

how does PhiX174 replicate

A

replicate via dsDNA intermediate and rolling circle (some genes overlap so there is frame reading)

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27
Q

outline PhiX174s genome

A

has 4 distinct intergenic region

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28
Q

how many intergenic regions does PhiX174 have

A

4

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29
Q

how are PhiX174 genes read

A

by frame reading due to overlapping genes

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30
Q

outline PhiX174s Life Cycle

A
  1. attachment to bacterial host followed by injection +ssDNA
  2. due to genomes positive polarity, DNA replication must commence before viral genes can be transcribed
  3. DNA replication in 3 stages - 1.ssDNA convert to dsDNA, 2.amplification of ds model
  4. ss genomic DNA synthesis and packaging
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31
Q

which type of DNA is like mRNA

A

-ssDNA is like mRNA

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32
Q

why cants +ssDNA be transcribed direcltlyq

A

wrong direction/sense/polarity

needs to be -ssDNA to be like mRNA - therefore have to replicate it first using host cell machinary

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33
Q

what are the three distinct stages of PhiX174 DNA replication

A
  1. ssDNA convert to dsDNA by going from +sense to -sense
  2. amplification of ds molecule
  3. ss genomic DNA synthesis and packaging
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34
Q

name some ssDNA bacteriophages

A

M13 (f1) are filamentous phages that infect E.coli throug pili and are able to produce new virions without lysing the host cell

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35
Q

name an phage that is able to produce virions without lysis host cell

A

M13 (f1) as filamentous and infect E.coli through pilli

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36
Q

what bacteria do M13/f1 viruses infect

A

E.coli

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37
Q

how do M13 phages infect bacteria

A

infect E.coli as they are filamentous and can use their pili to produce new virions without lysing the cell

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38
Q

outline genome of M13/f1 phage

A

circular, ssDNA - 6.4kb long

10 genes in genome

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39
Q

name a filamentous phage

A

m13/f1 it has 6.4 kb genome with 10genes in genome

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40
Q

how many genes in M13 phage

A

10

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41
Q

what genes are included in M13 phage

A

phage DNA synthesis genes
capsid structure genes
assembly genes

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42
Q

what are the basic genes every phage needs in its genome

A

phage DNA synthesis genes
capsid structure genes
phage assembly genes

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43
Q

what phage is often used for sequencing

A

M13 phage

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44
Q

outline life cycle of m13 phage

A

phage particle binds to F pilus = only infects F+, HFr and F cells
ssDNA enters cells designated as +strand
rolling circle replication, + strands are packaged in phage and coat proteins
exit cell as phage particle

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45
Q

name a temperate phage

A

lamda phage

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46
Q

what lamda phage - temperate or virulent

A

temperate

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47
Q

outline lifecycle of lamda phage

A

lamda tail binds to LamB protein on bacteria
Lamda ejects DNA
left end of lamda DNa is put into the capsid first and when the lamda DNA comes out of the phage head, the right end exits first
only takes 45mins from virus adsorption for cell lysis to occur, releasing the phages during lytic cycle: very quick
consists of an eclipse period, where intracellular accumulation phase and the lysis and release phase occurs

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48
Q

how long from DNA ejection does it take lamda phages to lyse the cell

A

45mins

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49
Q

what phases of phage infection exist

A

eclipse phase, intracellular accumulation phase, then lysis

then release phase

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50
Q

name a phage that has an extremely quick cycle

A

bacteriophage

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51
Q

how is lytic phase in lamda phage induced

A

by stressors

a prophage occasionally exits the bacterial chromosome,initiating a lytic cycle

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52
Q

in lamda phage, what is induction associated with

A

the cleavage of CI repressor, producing Cro protein which inhibits further production of CI

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53
Q

how does lamda phage bind to bacteria

A

lamda tail binds to LamB protein on host cell

54
Q

how many gene products does T4 phage encode

A

300

55
Q

does T4phage encode an RNAP

A

no -phage encoded proteins do sequentially modify host RNAP though

56
Q

what is bp number of bacteriophage T4

A

169kbp

57
Q

outline structure of T4 phage

A

long tail fibres with ravelled up short tailed fibres
upon binding base plate changes conformation and tail sheet extracts, causing GP5 @ end of tail tube to puncture outer membrane of the cell
lysosome domain activated, degrading periplasm
DNA from head of phage travels through tail tube and enters Ecoli

58
Q

which phage has a tail fibre AND base plate

A

T4 bacteriophage

59
Q

which base plate has genome encoding c/300 gene products

A

T4 bacteriophage

60
Q

outline conformational changes that a T4 phage undergoes to eject its DNA

A

long tail fibres with ravelled up short tail fibres
upon binding base plate changes conformation and tail sheet contracts, causing the GP5 @ end of tail tube to puncture outermembrane of the cell = lysosome domain activated, degrading periplasm - DNA travels through tail tube eand enters E.coli

61
Q

what is DNA type of T4

A

dsDNA linear enclosed in capsid and attached to tail

62
Q

when base plate of T4 changes conformation, what happens

A

tail sheet contracts, causing GP5 @ end of tail tube to puncture outermembran
activates lysosome domain
degrades perimplasm
DNA travels down tube into cell

63
Q

is T4 temperate or virulent

A

virulent

64
Q

outline life cycle of t4

A
  1. attachment
  2. entry phage DNA & degrade hostDNA
  3. synthesis viral genomes+proteins
  4. assembly of head,tails and tail fibres –>RELEASe
65
Q

what components are there to a t4

A

head
tails
tail fibres

66
Q

compare DNA structure of T4 - lamda - M13

A

T4 - linear dsDNA enclosed in capsid and attached to tail
lamda - linear dsDNA
M13 - +ssDNA circular

67
Q

compare gfenome size of T4 - lamda - M13

A

T4 - 172kb
lamda - 48.5kb
M13 - 6kb

68
Q

which phage has largest genome size

A

T4

69
Q

compare structure T4 - lamda - M13

A

T4 - capsid, head, tail, base plates (head, collar tail)
lamda - head and tail
M13 - filamentous

70
Q

outline different cycles in T4 - lamda - M13

A

T4 - virulent
M13 - can produce new virion without lysing host cell
lamda - temperate

71
Q

name order and family of T4 phage

A

order - caudovirales

family - miyoviridae

72
Q

outline main bacteria and bacteriophage arms race

A

RM restriction modification defense system

73
Q

what is RM - where found

A

restriction modification defense system found in bacteria

74
Q

why has RM evolved

A

due to bacteria and phage arms races

75
Q

outline a method of bacteria to try and destroy phage

A

have restriction endonucleases which restrict phage DNA through cleaving - restrictoin system to protenct themselves

76
Q

how do bacteria destroy phage DNa

A

host DNA methylated, so any foreign DNA is noticed and cleaved - bacteria

77
Q

give an example of how phages evade restriction

A

some viruses encode their own methyltransferase to evade RM system

78
Q

how do phages overcome RM system

A

they encode their own methyltransferase

79
Q

give an example of a bacterial “immune system”

A

CRISPR-Cas mechanism

80
Q

what are the three main microbial defense mechanisms against phage

A

the restriction-modification system,
CRISPR (clustered regularly interspersed palindromic repeats) loci together with their associated cas genes, and the abortive infection system

81
Q

in how many bacteria is the restriction modification system present in

A

over 90% of sequenced bacterial and archaeal genomes

82
Q

what are the two activities of the RM system

A

one that restricts incoming foreign genetic material

one that protects host genetic material from restriction

83
Q

what are RM activities mediated by

A

the recognition of a sspecific DNA sequence on average 4-8 bp long

84
Q

how long is the DNA sequence that mediates RM system

A

4-8bo

85
Q

how is protection conferred in the RM system

A

methylation of specific bases in the recognition sequence in host genome

86
Q

what happens to non-methylated DNA in bacteria

A

foreign therefore cleaved

87
Q

what does the minimal composition of RM systems consist of

A

methyltransferase gene that performs the defense activity and a restriction endonuclease gene that performs the foreign restriction activity

88
Q

what serves as the basis for identification of RM systems in newly sequenced genomes

A

the methyltransferase

89
Q

why does the methyltransferase serve as the basis for RM system identification and not the restriction endonuclease?

A

because the restriction endonuclease undergoes rapid evolution

90
Q

what does methyltransferase in RM do

A

performs defense activity of bacteria by methylating its DNA

91
Q

what does restriction endonuclease do in RM

A

recognizes foreign unmethylated DNA and cleaves it

92
Q

which enzyme in RM recognises foreign phage DNA a

A

restriction endonuclease

93
Q

which enzyme performs defense activity in RM

A

methyltransferase

94
Q

how can phages evade RM systems

A

some acquired methyltransferase
some stimulate host MTase so it confers protection to phage genomes
some code for proteins that target+shut down REase

95
Q

give an example of bacterial acquired adaptive immunity

A

CRISPR/Cas system

96
Q

what is CRISPR/Cas system an example of

A

bacterial acquired adaptive immunity

97
Q

where is CRISPR/Cas found

A

in E.coli

98
Q

what is CRISPR/Cas composed of

A

a unique nucleotide arrangement in E.coli - cluster of direct repeats interspersed with variable sequences (spaces - around 30bp long)
associated with other genes - Cas genes

99
Q

what does CRISPR stand for

A

clusters of regularly interspersed short palindromic repeats

100
Q

what happens to CRISPRs

A

theyre transcribed and processed into short RNAs that function in RNAinterference

101
Q

what genes are CRISPRs associated with

A

Cas genes - code for a cascade

102
Q

what does a CRISPR locus contain

A

repeating sequences which are identiacal

variable sequences called spacers which are acquired from foreign genetic material like viruses/plasmids

103
Q

what are spacers

A

variable sequences found at CRISPR locus which are acquired from foreign genetic material like viruses/plasmids

104
Q

what three stages of CRISPR/Cas mechanisms are there

A
  1. spacer acquisition: allow future immunization to memorize invader
  2. expression and procession - of CRISPR repeats 0 spacer array is transcribed into a long primary RNA transcript
  3. interference or silencing of phage DNA - immunity
105
Q

what do RNAse and Cas3 do to incoming phage DNA

A

bind and destroy it

106
Q

which molecules bind to incoming phage DNA and destroy it

A

RNAse and Cas3

107
Q

what is abortive infection aka

A

cellular suicide

108
Q

when does abortive infection occur

A

if a phage has successfully entered host cell and avoided restriction by host RM systems and by CRISPR

109
Q

what does abortive infection describe

A

a collective term describing host mechanisms that interrupt with phage development at different stages of phage transcription, genome replication and phage packaging

110
Q

what method is a host mechanism that interrupts phage development, phage genome replication and transcription and phage packaging

A

abortive infection

111
Q

what mechanism do host cells resort to if RM or CRISPR/Cas has not occured

A

abortive infection: cell suicide

112
Q

why is abortive infection advantageous

A

it confines the infection to the sacrificed cell and prevents the spread of infection to surrounding cells

113
Q

what effect do Abi genes have

A

they have a toxic effect on their host

114
Q

what is the CRISPR locus in E.coli transcribed into

A

short RNAs that function in RNA interference

115
Q

what is RNAi

A

RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.

116
Q

what RNA inhibits gene expression - typically by causing the destruction of specific mRNA molecules

A

RNAinterferenfce

117
Q

how can lysogenic phages cause disease

A

they carry genes that can enhance the virulence of the bacterial host - eg. encode toxins - so once these genes are inegrated into the bacterial chroms, they can cause release of potent disease causing toins

118
Q

outline the associated phage, gene product and phenotype of Vibrio cholerae

A

phage - CTX phage
product-cholerae toxin
phenotype- cholera

119
Q

outline the associated phage, gene product and phenotype of e.coli

A

lamda phage
shigalike toxin
hemorrhagic diarrhea

120
Q

outline the associated phage, gene product and phenotype of Clostridium botulinum

A

clostridial phages
botulinum toxin
botulism -food poisoning

121
Q

outline the associated phage, gene product and phenotype of Corynebacterium diphtheriae

A

corynephage beta
diphtheria toxin
diphtheria

122
Q

outline the associated phage, gene product and phenotype of Streptococcus pyogenes

A

T12
erythrogenic toxins
scarlet fever

123
Q

which phage associated with cholera

A

CTX phage

124
Q

which phage associated with hemorrhagic diarrhea

A

lamda

125
Q

which phage associated with botulism - food poisoning

A

clostridial phage

126
Q

which phage associated with diphtheria

A

diphtheria toxin

127
Q

which phage associated with scarlet fever

A

T12

128
Q

what is phage display

A

laboratory technique to study protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them.

129
Q

what are three steps to phage display technique

A
  1. antibody library construction and display onto phage surface
  2. selection by pinning the library against antigen (Ag) targets
  3. screening for desired specificity
130
Q

in phage display - how is an antibody library constructed

A

a gene encoding a protein of interest is inderted into a phage coat protein gene, causeing the phage to display protein on outside with gene insideq

HELENP CBG VIROLOGY AND IMMUNITY (8 decks)

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