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
2
Q
what do you call an obligate intracellular parasite that multiplies by making use of the host cell’s biosynthetic machinary
A
bacteriophage
3
Q
what is the significance for using bacteriophages
A
models for animal cell viruses
gene transfer in bacteria
medical application
lysogenic conversion
4
Q
name some uses for bacteriophages
A
models for animal cell viruses
gene transfer in bacteria
medical application
lysogenic conversion
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
6
Q
give an example where phage therapy has been used
A
to treat dysentery
7
Q
when phage attaches to host cell and injects DNA, what 2 pathways can happen
A
it can be lytic or lysogenic
8
Q
what is a lytic phage aka
A
virulent
9
Q
what is a virulent phage aka
A
lytic
10
Q
what is a temperate phage aka
A
lysogenic
11
Q
what is a lysogenic phage aka
A
temperate
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
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
14
Q
what is a phage that can only kill by multiplying within host cell then lysing called
A
lytic phage
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
16
Q
what happens to the expression of most phage genes in the lysogenic phage
A
expression of most genes is repressed - -inactive
17
Q
what is genome of a phage
A
DNA or RNA
18
Q
what triggers conversion of lysogenic phage to lytic
A
stressor
eg. UV- host excises its virus genome making it active
19
Q
how do phages attach to host cell
A
bind to specific receptors that are proteins or carbs in the bac cell wall
20
Q
what is PhiX174
A
extensively studied virus = isometric with 20 traingular faces
microvirus
21
Q
give an example of a microvirus
A
PhiX174
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
23
Q
how many nucleotides is PhiX174
A
5386 nucleotides - have a very small genome
24
Q
what type genome does PhiX174 have
A
plus sense ssDNA - useful for studying DNA replication
25
what was the fist viral genome to be sequenced
PhiX174
26
how does PhiX174 replicate
replicate via dsDNA intermediate and rolling circle (some genes overlap so there is frame reading)
27
outline PhiX174s genome
has 4 distinct intergenic region
28
how many intergenic regions does PhiX174 have
4
29
how are PhiX174 genes read
by frame reading due to overlapping genes
30
outline PhiX174s Life Cycle
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
3. ss genomic DNA synthesis and packaging
31
which type of DNA is like mRNA
-ssDNA is like mRNA
32
why cants +ssDNA be transcribed direcltlyq
wrong direction/sense/polarity
| needs to be -ssDNA to be like mRNA - therefore have to replicate it first using host cell machinary
33
what are the three distinct stages of PhiX174 DNA replication
1. ssDNA convert to dsDNA by going from +sense to -sense
2. amplification of ds molecule
3. ss genomic DNA synthesis and packaging
34
name some ssDNA bacteriophages
M13 (f1) are filamentous phages that infect E.coli throug pili and are able to produce new virions without lysing the host cell
35
name an phage that is able to produce virions without lysis host cell
M13 (f1) as filamentous and infect E.coli through pilli
36
what bacteria do M13/f1 viruses infect
E.coli
37
how do M13 phages infect bacteria
infect E.coli as they are filamentous and can use their pili to produce new virions without lysing the cell
38
outline genome of M13/f1 phage
circular, ssDNA - 6.4kb long
| 10 genes in genome
39
name a filamentous phage
m13/f1 it has 6.4 kb genome with 10genes in genome
40
how many genes in M13 phage
10
41
what genes are included in M13 phage
phage DNA synthesis genes
capsid structure genes
assembly genes
42
what are the basic genes every phage needs in its genome
phage DNA synthesis genes
capsid structure genes
phage assembly genes
43
what phage is often used for sequencing
M13 phage
44
outline life cycle of m13 phage
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
45
name a temperate phage
lamda phage
46
what lamda phage - temperate or virulent
temperate
47
outline lifecycle of lamda phage
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
48
how long from DNA ejection does it take lamda phages to lyse the cell
45mins
49
what phases of phage infection exist
eclipse phase, intracellular accumulation phase, then lysis
| then release phase
50
name a phage that has an extremely quick cycle
bacteriophage
51
how is lytic phase in lamda phage induced
by stressors
| a prophage occasionally exits the bacterial chromosome,initiating a lytic cycle
52
in lamda phage, what is induction associated with
the cleavage of CI repressor, producing Cro protein which inhibits further production of CI
53
how does lamda phage bind to bacteria
lamda tail binds to LamB protein on host cell
54
how many gene products does T4 phage encode
300
55
does T4phage encode an RNAP
no -phage encoded proteins do sequentially modify host RNAP though
56
what is bp number of bacteriophage T4
169kbp
57
outline structure of T4 phage
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
which phage has a tail fibre AND base plate
T4 bacteriophage
59
which base plate has genome encoding c/300 gene products
T4 bacteriophage
60
outline conformational changes that a T4 phage undergoes to eject its DNA
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
what is DNA type of T4
dsDNA linear enclosed in capsid and attached to tail
62
when base plate of T4 changes conformation, what happens
tail sheet contracts, causing GP5 @ end of tail tube to puncture outermembran
activates lysosome domain
degrades perimplasm
DNA travels down tube into cell
63
is T4 temperate or virulent
virulent
64
outline life cycle of t4
1. attachment
2. entry phage DNA & degrade hostDNA
3. synthesis viral genomes+proteins
4. assembly of head,tails and tail fibres -->RELEASe
65
what components are there to a t4
head
tails
tail fibres
66
compare DNA structure of T4 - lamda - M13
T4 - linear dsDNA enclosed in capsid and attached to tail
lamda - linear dsDNA
M13 - +ssDNA circular
67
compare gfenome size of T4 - lamda - M13
T4 - 172kb
lamda - 48.5kb
M13 - 6kb
68
which phage has largest genome size
T4
69
compare structure T4 - lamda - M13
T4 - capsid, head, tail, base plates (head, collar tail)
lamda - head and tail
M13 - filamentous
70
outline different cycles in T4 - lamda - M13
T4 - virulent
M13 - can produce new virion without lysing host cell
lamda - temperate
71
name order and family of T4 phage
order - caudovirales
| family - miyoviridae
72
outline main bacteria and bacteriophage arms race
RM restriction modification defense system
73
what is RM - where found
restriction modification defense system found in bacteria
74
why has RM evolved
due to bacteria and phage arms races
75
outline a method of bacteria to try and destroy phage
have restriction endonucleases which restrict phage DNA through cleaving - restrictoin system to protenct themselves
76
how do bacteria destroy phage DNa
host DNA methylated, so any foreign DNA is noticed and cleaved - bacteria
77
give an example of how phages evade restriction
some viruses encode their own methyltransferase to evade RM system
78
how do phages overcome RM system
they encode their own methyltransferase
79
give an example of a bacterial "immune system"
CRISPR-Cas mechanism
80
what are the three main microbial defense mechanisms against phage
the restriction-modification system,
CRISPR (clustered regularly interspersed palindromic repeats) loci together with their associated cas genes, and the abortive infection system
81
in how many bacteria is the restriction modification system present in
over 90% of sequenced bacterial and archaeal genomes
82
what are the two activities of the RM system
one that restricts incoming foreign genetic material
| one that protects host genetic material from restriction
83
what are RM activities mediated by
the recognition of a sspecific DNA sequence on average 4-8 bp long
84
how long is the DNA sequence that mediates RM system
4-8bo
85
how is protection conferred in the RM system
methylation of specific bases in the recognition sequence in host genome
86
what happens to non-methylated DNA in bacteria
foreign therefore cleaved
87
what does the minimal composition of RM systems consist of
methyltransferase gene that performs the defense activity and a restriction endonuclease gene that performs the foreign restriction activity
88
what serves as the basis for identification of RM systems in newly sequenced genomes
the methyltransferase
89
why does the methyltransferase serve as the basis for RM system identification and not the restriction endonuclease?
because the restriction endonuclease undergoes rapid evolution
90
what does methyltransferase in RM do
performs defense activity of bacteria by methylating its DNA
91
what does restriction endonuclease do in RM
recognizes foreign unmethylated DNA and cleaves it
92
which enzyme in RM recognises foreign phage DNA a
restriction endonuclease
93
which enzyme performs defense activity in RM
methyltransferase
94
how can phages evade RM systems
some acquired methyltransferase
some stimulate host MTase so it confers protection to phage genomes
some code for proteins that target+shut down REase
95
give an example of bacterial acquired adaptive immunity
CRISPR/Cas system
96
what is CRISPR/Cas system an example of
bacterial acquired adaptive immunity
97
where is CRISPR/Cas found
in E.coli
98
what is CRISPR/Cas composed of
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
what does CRISPR stand for
clusters of regularly interspersed short palindromic repeats
100
what happens to CRISPRs
theyre transcribed and processed into short RNAs that function in RNAinterference
101
what genes are CRISPRs associated with
Cas genes - code for a cascade
102
what does a CRISPR locus contain
repeating sequences which are identiacal
| variable sequences called spacers which are acquired from foreign genetic material like viruses/plasmids
103
what are spacers
variable sequences found at CRISPR locus which are acquired from foreign genetic material like viruses/plasmids
104
what three stages of CRISPR/Cas mechanisms are there
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
what do RNAse and Cas3 do to incoming phage DNA
bind and destroy it
106
which molecules bind to incoming phage DNA and destroy it
RNAse and Cas3
107
what is abortive infection aka
cellular suicide
108
when does abortive infection occur
if a phage has successfully entered host cell and avoided restriction by host RM systems and by CRISPR
109
what does abortive infection describe
a collective term describing host mechanisms that interrupt with phage development at different stages of phage transcription, genome replication and phage packaging
110
what method is a host mechanism that interrupts phage development, phage genome replication and transcription and phage packaging
abortive infection
111
what mechanism do host cells resort to if RM or CRISPR/Cas has not occured
abortive infection: cell suicide
112
why is abortive infection advantageous
it confines the infection to the sacrificed cell and prevents the spread of infection to surrounding cells
113
what effect do Abi genes have
they have a toxic effect on their host
114
what is the CRISPR locus in E.coli transcribed into
short RNAs that function in RNA interference
115
what is RNAi
RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.
116
what RNA inhibits gene expression - typically by causing the destruction of specific mRNA molecules
RNAinterferenfce
117
how can lysogenic phages cause disease
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
outline the associated phage, gene product and phenotype of Vibrio cholerae
phage - CTX phage
product-cholerae toxin
phenotype- cholera
119
outline the associated phage, gene product and phenotype of e.coli
lamda phage
shigalike toxin
hemorrhagic diarrhea
120
outline the associated phage, gene product and phenotype of Clostridium botulinum
clostridial phages
botulinum toxin
botulism -food poisoning
121
outline the associated phage, gene product and phenotype of Corynebacterium diphtheriae
corynephage beta
diphtheria toxin
diphtheria
122
outline the associated phage, gene product and phenotype of Streptococcus pyogenes
T12
erythrogenic toxins
scarlet fever
123
which phage associated with cholera
CTX phage
124
which phage associated with hemorrhagic diarrhea
lamda
125
which phage associated with botulism - food poisoning
clostridial phage
126
which phage associated with diphtheria
diphtheria toxin
127
which phage associated with scarlet fever
T12
128
what is phage display
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
what are three steps to phage display technique
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
in phage display - how is an antibody library constructed
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
