Bacterial viology Flashcards
define bacteriophage
obligate intracellular parasites that multiply inside bacteria making use of some or all of the host biosynthetic machinery
what can bacteriophage genomes be? and where do bacteriophages bind to?
DNA or RNA both single or double stranded
they bind to specific receptors that are proteins or carbohydrates in the bacterial cell wall
Why is cholera dangerous?
injection of bacteriophage causes protein to open up transmembrane protein and this causes diarrhea
what is PhiX174? who discovered it? why was it significant? how big is its genome?
Fred Sanger. Extensively studied isometric virus with triangular faces. first DNA based genome to be sequenced -5386 nucleotides. Very small genome which is infectious by itself. Models for fundamentals of DNA replication. +sense ssDNA circular. 4 distinct intergenic regions.
lytic /virulent phage?
PHAGE THAT CAN ONLY MULTIPLY WITHIN BACTERIA AND KILL THE CELL BY LYSIS
lysogenc/ temperate phage?
phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. Expression of most phage genes repressed as prophage.
define prophage:
the genetic material of a bacteriophage, incorporated into the genome of a bacterium and able to produce phages if specifically activated.
life cycle of PhiX174:
1) attachment of virus to the bacterial host folowed by injection of ssDNA
2) due to genomes’ positive polarity,DNA replication must commence before viral genes can be transcribed
3) DNA replication
- a)ssDNA converted to dsDNA
- b)amplification of the ds molecule
- c)ss genomic DNA synthesis and packaging
What are m13/f1 phages?
ssDNa bacteriphages
describe M13
filamentous phages that infect e.coli through pili, and are able to produce new virions without lysing the host cell.
what are key M13 structural elements wrt genome organisation and size etc?
Circular, single-stranded DNA
~6.4 kb long
10 genes in the genome.
why study bacteriophages?
Models for animal cell viruses
Gene transfer in bacteria
Medical applications
Lysogenic conversion
can bacteriophages infect archaea?
yes
how do bacteriophages infect cells?
Bind to specific receptors that are proteins or carbohydrates in the bacterial cell wall
what type of virus is PhiX174?
a microvirus
what is ds lamda bacteriophage replication cycle like? give specific times
very quick during lytic stage.
0mins-viruses absorb on lambda
30mins-viruses reproduced in cell
45mins-cell membrane breaks and releases phages
what does the lamda cycle consist of?
eclipse period,intracellular accumulation phase, lysis and release phase
what does the lamda lysogenic cycle consist of? (5)
1) phage DNA cirularizes (middle of both cycles)
2) phage DNA integrates into the bacterial chromosome this becomes a prophage
3) the bacterium reproduces normally and the prophage is copied too and transmitted to daughter cells
4) many cell divisions produce a colony of bacteria infected with prophages
5) occassionally the prophage exits the bacterial chromosome and this can initiate a lytic cycle.
what does the lamda lysogenic cycle consist of? (5)
1) phage DNA circularises (middle of both cycles)
2) new phage DNA and proteins are synthesized and assembled into phages
3) cell lyses releasing phages
4) phage attches itsef to new host bacterium cell and injects its DNA
5) sometimes phage integrates into bacterial chromosome becoming a prophage and entering lysogenic cycle
what is bacteriophage T4 made up of structurally?
head,collar,tail,base plate
does the T4 bacteriophage encode an RNAP?
no. however it does encode proteins that modify host RNAP
how long is T4 bacteriophage genome and how many products does it encode?
170,000bp and 300 gene products
what system do bacteria use against bacteriophages?
a restriction system
how does the bacterial restriction system work?
1) Entering DNA is methylated by modification enzyke and made resistant to degradation by restriction endonucleases
2) Restriction endonuclease can then cleave foreign DNA before it has a chance to be methylate
how have some bacteriophages overcome the restriction system?
some modify their DNA. Uncut genome is then protected by methylation. Bacteriophage then replicates and eventually this leads to cell breakage and cell death
Bacterial restriction system DETAIL
Bacteria have restriction endonucleases, which cleave double stranded DNA at specific points into fragments, which are then degraded further by other endonucleases.
This prevents infection by destroying the foreign DNA introduced by an infectious agent (such as a bacteriophage).
Given that the sequences of the restriction enzymes recognize are very short, the bacterium itself will almost certainly have many of these sequences present in its own DNA.
In order to prevent destruction of its own DNA by the restriction enzymes, the bacterium marks its own DNA by adding methyl groups to it. This modification must not interfere with the DNA base-pairing, and therefore, usually only a few specific bases are modified on each strand.
Endonucleases cleave internal/non-terminal phosphodiester bonds. Restriction endonucleases cleave internal phosphodiester bonds only after recognising specific sequences in DNA which are usually 4-6 base pairs long, and often palindromic
what are CRISPR’s
Clusters of regularly interspersed short palindromic repeats in prokaryotes that are transcribed and processed into short RNAs that function in RNA interference
what is the the CRISPR locus in E. coli transcribed to?
The CRISPR locus in E. coli is transcribed into a larger precursor RNA
what organisms insert spacer sequences as a form of generating immunological memory
Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown.
what do bacteria and archaea insert into CRISPR loci to generate immunological memory?
spacer sequences
what mediates spacer acquisition in e.coli in vivo?
Cas1–Cas2
what bacterium gene products to vibrio choler,e.coli and clostridium botulinum all produce?
toxins
what bacterium has a CTX phage that infects it that produces a cholera phenotype?
vibrio cholera bacterium
what can e.coli by infected by that causes hemorrhagic diarrhea?
lambder phage
what phage can infect clostridium botulinum and what does it cause?
clostridial phage and causes botulism (food poisoning)
what does the clostridial phage infect?
clostridium botulinum
what are the 3 steps to phage display?
1) antibody library construction and display onto phage surface
2) selection by panning the library against Ag targets
3) screen for the desired specificty
Human antibody library steps:
1) isolate B cells
2) PcR Vh and Vl genes
3) Display hmAbs fragments
4) visible on phage
library selection steps:
1) Ag binding
2) epitopes, washing
3) elution –> amplification sometime
4) screening
5) cloning
6) antigens
define antigen
a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies.
define epitope
the part of an antigen molecule to which an antibody attaches itself
