CBG Lecture 24: Bacteriophage Flashcards
deine bacteriophage
obligate intracellular parasites that multiply inside bacteria by making use of some or all of host biosynthetic machinary
what do you call an obligate intracellular parasite that multiplies by making use of the host cell’s biosynthetic machinary
bacteriophage
what is the significance for using bacteriophages
models for animal cell viruses
gene transfer in bacteria
medical application
lysogenic conversion
name some uses for bacteriophages
models for animal cell viruses
gene transfer in bacteria
medical application
lysogenic conversion
what is phage therapy
in Eastern Europe: can order bacteriophages to treat infection: no problem with resistance and its a form of autodosing
phages can treat dysentery
give an example where phage therapy has been used
to treat dysentery
when phage attaches to host cell and injects DNA, what 2 pathways can happen
it can be lytic or lysogenic
what is a lytic phage aka
virulent
what is a virulent phage aka
lytic
what is a temperate phage aka
lysogenic
what is a lysogenic phage aka
temperate
what is a lytic phage
phage that can only kill the host cell by multiplication within it, then killing the cell by lysis
what is a lysogenic phage
can either multiply by lytic cycle or enter a dormant state where the expression of most phage genes are repressed, in a prophage
what is a phage that can only kill by multiplying within host cell then lysing called
lytic phage
what is a phage that can either multiply by lytic cycle or remain inactive and dormant with its genes repressed in a prophage called
lysogenic phage
what happens to the expression of most phage genes in the lysogenic phage
expression of most genes is repressed - -inactive
what is genome of a phage
DNA or RNA
what triggers conversion of lysogenic phage to lytic
stressor
eg. UV- host excises its virus genome making it active
how do phages attach to host cell
bind to specific receptors that are proteins or carbs in the bac cell wall
what is PhiX174
extensively studied virus = isometric with 20 traingular faces
microvirus
give an example of a microvirus
PhiX174
why is PhiX174 historically important
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
how many nucleotides is PhiX174
5386 nucleotides - have a very small genome
what type genome does PhiX174 have
plus sense ssDNA - useful for studying DNA replication
what was the fist viral genome to be sequenced
PhiX174
how does PhiX174 replicate
replicate via dsDNA intermediate and rolling circle (some genes overlap so there is frame reading)
outline PhiX174s genome
has 4 distinct intergenic region
how many intergenic regions does PhiX174 have
4
how are PhiX174 genes read
by frame reading due to overlapping genes
outline PhiX174s Life Cycle
- attachment to bacterial host followed by injection +ssDNA
- due to genomes positive polarity, DNA replication must commence before viral genes can be transcribed
- DNA replication in 3 stages - 1.ssDNA convert to dsDNA, 2.amplification of ds model
- ss genomic DNA synthesis and packaging
which type of DNA is like mRNA
-ssDNA is like mRNA
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
what are the three distinct stages of PhiX174 DNA replication
- ssDNA convert to dsDNA by going from +sense to -sense
- amplification of ds molecule
- ss genomic DNA synthesis and packaging
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
name an phage that is able to produce virions without lysis host cell
M13 (f1) as filamentous and infect E.coli through pilli
what bacteria do M13/f1 viruses infect
E.coli
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
outline genome of M13/f1 phage
circular, ssDNA - 6.4kb long
10 genes in genome
name a filamentous phage
m13/f1 it has 6.4 kb genome with 10genes in genome
how many genes in M13 phage
10
what genes are included in M13 phage
phage DNA synthesis genes
capsid structure genes
assembly genes
what are the basic genes every phage needs in its genome
phage DNA synthesis genes
capsid structure genes
phage assembly genes
what phage is often used for sequencing
M13 phage
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
name a temperate phage
lamda phage
what lamda phage - temperate or virulent
temperate
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
how long from DNA ejection does it take lamda phages to lyse the cell
45mins
what phases of phage infection exist
eclipse phase, intracellular accumulation phase, then lysis
then release phase
name a phage that has an extremely quick cycle
bacteriophage
how is lytic phase in lamda phage induced
by stressors
a prophage occasionally exits the bacterial chromosome,initiating a lytic cycle
in lamda phage, what is induction associated with
the cleavage of CI repressor, producing Cro protein which inhibits further production of CI
how does lamda phage bind to bacteria
lamda tail binds to LamB protein on host cell
how many gene products does T4 phage encode
300
does T4phage encode an RNAP
no -phage encoded proteins do sequentially modify host RNAP though
what is bp number of bacteriophage T4
169kbp
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
which phage has a tail fibre AND base plate
T4 bacteriophage
which base plate has genome encoding c/300 gene products
T4 bacteriophage
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
what is DNA type of T4
dsDNA linear enclosed in capsid and attached to tail
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
is T4 temperate or virulent
virulent
outline life cycle of t4
- attachment
- entry phage DNA & degrade hostDNA
- synthesis viral genomes+proteins
- assembly of head,tails and tail fibres –>RELEASe
what components are there to a t4
head
tails
tail fibres
compare DNA structure of T4 - lamda - M13
T4 - linear dsDNA enclosed in capsid and attached to tail
lamda - linear dsDNA
M13 - +ssDNA circular
compare gfenome size of T4 - lamda - M13
T4 - 172kb
lamda - 48.5kb
M13 - 6kb
which phage has largest genome size
T4
compare structure T4 - lamda - M13
T4 - capsid, head, tail, base plates (head, collar tail)
lamda - head and tail
M13 - filamentous
outline different cycles in T4 - lamda - M13
T4 - virulent
M13 - can produce new virion without lysing host cell
lamda - temperate
name order and family of T4 phage
order - caudovirales
family - miyoviridae
outline main bacteria and bacteriophage arms race
RM restriction modification defense system
what is RM - where found
restriction modification defense system found in bacteria
why has RM evolved
due to bacteria and phage arms races
outline a method of bacteria to try and destroy phage
have restriction endonucleases which restrict phage DNA through cleaving - restrictoin system to protenct themselves
how do bacteria destroy phage DNa
host DNA methylated, so any foreign DNA is noticed and cleaved - bacteria
give an example of how phages evade restriction
some viruses encode their own methyltransferase to evade RM system
how do phages overcome RM system
they encode their own methyltransferase
give an example of a bacterial “immune system”
CRISPR-Cas mechanism
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
in how many bacteria is the restriction modification system present in
over 90% of sequenced bacterial and archaeal genomes
what are the two activities of the RM system
one that restricts incoming foreign genetic material
one that protects host genetic material from restriction
what are RM activities mediated by
the recognition of a sspecific DNA sequence on average 4-8 bp long
how long is the DNA sequence that mediates RM system
4-8bo
how is protection conferred in the RM system
methylation of specific bases in the recognition sequence in host genome
what happens to non-methylated DNA in bacteria
foreign therefore cleaved
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
what serves as the basis for identification of RM systems in newly sequenced genomes
the methyltransferase
why does the methyltransferase serve as the basis for RM system identification and not the restriction endonuclease?
because the restriction endonuclease undergoes rapid evolution
what does methyltransferase in RM do
performs defense activity of bacteria by methylating its DNA
what does restriction endonuclease do in RM
recognizes foreign unmethylated DNA and cleaves it
which enzyme in RM recognises foreign phage DNA a
restriction endonuclease
which enzyme performs defense activity in RM
methyltransferase
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
give an example of bacterial acquired adaptive immunity
CRISPR/Cas system
what is CRISPR/Cas system an example of
bacterial acquired adaptive immunity
where is CRISPR/Cas found
in E.coli
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
what does CRISPR stand for
clusters of regularly interspersed short palindromic repeats
what happens to CRISPRs
theyre transcribed and processed into short RNAs that function in RNAinterference
what genes are CRISPRs associated with
Cas genes - code for a cascade
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
what are spacers
variable sequences found at CRISPR locus which are acquired from foreign genetic material like viruses/plasmids
what three stages of CRISPR/Cas mechanisms are there
- spacer acquisition: allow future immunization to memorize invader
- expression and procession - of CRISPR repeats 0 spacer array is transcribed into a long primary RNA transcript
- interference or silencing of phage DNA - immunity
what do RNAse and Cas3 do to incoming phage DNA
bind and destroy it
which molecules bind to incoming phage DNA and destroy it
RNAse and Cas3
what is abortive infection aka
cellular suicide
when does abortive infection occur
if a phage has successfully entered host cell and avoided restriction by host RM systems and by CRISPR
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
what method is a host mechanism that interrupts phage development, phage genome replication and transcription and phage packaging
abortive infection
what mechanism do host cells resort to if RM or CRISPR/Cas has not occured
abortive infection: cell suicide
why is abortive infection advantageous
it confines the infection to the sacrificed cell and prevents the spread of infection to surrounding cells
what effect do Abi genes have
they have a toxic effect on their host
what is the CRISPR locus in E.coli transcribed into
short RNAs that function in RNA interference
what is RNAi
RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.
what RNA inhibits gene expression - typically by causing the destruction of specific mRNA molecules
RNAinterferenfce
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
outline the associated phage, gene product and phenotype of Vibrio cholerae
phage - CTX phage
product-cholerae toxin
phenotype- cholera
outline the associated phage, gene product and phenotype of e.coli
lamda phage
shigalike toxin
hemorrhagic diarrhea
outline the associated phage, gene product and phenotype of Clostridium botulinum
clostridial phages
botulinum toxin
botulism -food poisoning
outline the associated phage, gene product and phenotype of Corynebacterium diphtheriae
corynephage beta
diphtheria toxin
diphtheria
outline the associated phage, gene product and phenotype of Streptococcus pyogenes
T12
erythrogenic toxins
scarlet fever
which phage associated with cholera
CTX phage
which phage associated with hemorrhagic diarrhea
lamda
which phage associated with botulism - food poisoning
clostridial phage
which phage associated with diphtheria
diphtheria toxin
which phage associated with scarlet fever
T12
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.
what are three steps to phage display technique
- antibody library construction and display onto phage surface
- selection by pinning the library against antigen (Ag) targets
- screening for desired specificity
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
