Download Bacteriophages: basic characteristics, biology and diversity.

Bacteriophages: basic characteristics,
biology and diversity.
Dr Mike Dyall-Smith, lab 3.07
[email protected]
M.D-S., 2006
What are bacteriophages?
Viruses of bacteria - first discovered 1915, Frederick Twort and but largely
researched by Felix D’Herelle
Bacteriophages (or phage):
are small (< 200 nm diam), acellular organisms that contain DNA or RNA
genomes enclosed in a protein coat.
are inactive outside cells, but once they have infected a cell they direct the cell
metabolism to assemble more virus particles.
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Lambda and P1: specialised or
site-specific recombination
Refs: Trun and Trempy, Fundamental Bacterial Genetics
Lewin, Genes VIII
Dr Mike Dyall-Smith, lab 3.07
[email protected]
M.D-S., 2006
Advantages of phages
Simple, small genome - easy to manipulate
Rapid and efficient infection of host cells
Rapid replication
Very high production rates and yields of virus
from host cells
Can clone foreign DNA into viral DNA
Can use their genes and enzymes or the virus
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Types of Infection
Lytic or Virulent - eg. T4. Virus has lysin gene and
destroys the host cell to liberate newly made virus
particles.
Temperate - eg. lambda, P1. Virus can maintain its
genome in a stable state (prophage) in the host cell for
an indefinite time, without lysing the host.
Chronic - eg. M13, fd. Once infected the host cell
continually produces virus particles at high rates,
slowing host cell growth and lowering viability.
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Temperate phage
Have a regulatory switch between lytic and temperate
states
When existing as a prophage, they express repressor
proteins to block expression of virus replication genes,
and also express genes to protect the cell from
superinfection by similar viruses (immunity genes)
Usually a small percentage of cells break out of the
prophage state and go through the lytic cycle.
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Temperate phage (eg. lambda)
From Prescott,
Harley and Klein,
2005
~49 kb
Phage lambda has linear, dsDNA has
ss , complementary 3’ overhangs
Once circularized, it can recombine with the host chromosome,
integrating into a specific site.
The virus genome is then almost completely inactive, but is
replicated along with the host cell chromosome.
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Lambda phage
From Prescott, Harley and Klein, 2005
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Specialised recombination
or site-specific recombination
recombination between specific sites that are not
necessarily completely homologous
lambda phage integrates into the host chromosome by
recombination at a specific (att) site
the lambda genome is excised from the E.coli
chromosome by recombination between the ends of the
prophage
lambda gene int codes for an integrase enzyme (Int
recombinase) that catalyses integration
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Specialised recombination
Extensive regions of homology are not required,
and Rec enzymes are not involved.
Can occur between two DNA molecules (lambda
integration), or within the same DNA molecule
(lambda excision)
Very energy efficient, ATP is not required for sitespecific recombination
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Specialised recombination
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Specialised recombination
lambda
E.coli chromosome
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Specialised recombination
If the att site is deleted from the E. coli
chromosome, integration is possible elsewhere,
but the efficiency is far less (<0.1%) compared
to the frequency of integration at att .
These ‘secondary attachment sites’, resemble
the authentic att sequences.
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Specialised recombination
attP = phage attachment site
attB = bacterial attachment site
- located between genes for
galactose utilization and biotin
synthesis.
attP and attB are also called
the POP’ and BOB’ sites, and
contain the same 15 nt core
sequence:
GCTTTTTTATACTAA
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Specialised recombination
Int recombinase catalyzes the
cutting within a 7 nt sequence
TTTATAC of the core sequence
• attB has two sites for Int
recombinase
• attP of lambda has five sites for
Int
• An E.coli factor, IHF (integration
host factor) is also bound.
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Specialised recombination
• IHF bends the flanking
sequences of attP core
sequence to be closer to the
core.
• Three IHF binding sites are in
attP. None are in attB.
• Once Int and IHF have bound,
and the two sites (attP and
attB) are close together, Int
catalyzes four phosphodiester
strand breakages.
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Lambda integration
The right end of the attP
core is joined to the left
end of the attB core and
vice versa
•Int catalyses the joins
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Lambda excision
•The integrated lambda
phage genome can be
excised by a reversal of the
same process.
•The process is called
excision
•Requires Int, IHF and a
second protein Xis
(excisionase)
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Site-Specific / specialised recombination
•Integrase acts in a similar way to type
I topoisomerases in that DNA strands
are broken one at a time
•However Int then joins the ends up
cross-wise (whereas topoisomerases
rejoin the same ends)
•Basic principle is that one Int enzyme
is required for each strand breakage
(so four are required)
•Int is a monomeric enzyme with an
active site capable of cutting or
ligating DNA
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Site-Specific / specialised recombination
•Two enzymes bound to each
recombination site
•At each site, only one enzyme cuts
the DNA, and the 3’ end is joined to a
Tyrosine
•The 5’-OH attacks the tyrosine bound
3’ end of an opposing molecule
forming a Holliday junction
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Site-Specific / specialised recombination
•Resolution occurs when the other
two enzyme molecules (not
involved in the first round) act on
the other pair of complementary
strands
•The result is a conservative strand
exchange, with no deletions or
insertions, and no use of ATP.
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Lambda recombination
Host protein IHF is also
required for both integration
and excision
IHF is a 20 kDa protein
Not essential for E.coli
Has the ability to wrap DNA on
a surface
IHF and Int bind at different
sequences within the core
sequence
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Excision of lambda
When Int and IHF bind to attP,
they generate a complex in
which all the binding sites are
brought together on the
surface of a protein
The complex is called an
INTASOME
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Intasome
When Int and IHF bind to attP,
they generate a complex in
which all the binding sites are
brought together on the
surface of a protein
The complex is called an
INTASOME
The current model suggests
that the attP intasome ‘traps’
attB
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Intasome
•The current model suggests
that the attP intasome ‘traps’
attB
•The initial reaction is not
based directly on att DNA
homology but by Int binding to
both att sites
•The two att sites are then
positioned on the intasome, in
the correct orientation for
recombination.
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Excision requires Xis
•In the integrated state,
the two ends are called
attL and attR.
•The lambda gene
product, Xis protein, is
required for excision, as
well as Int and IHF
•Why would a specific
phage factor be needed
for excision?
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Lambda
• Lambda phage
integration/excision
requires host factor and
two phage proteins
(Int,Xis)
• This means the system
cannot easily used in
other cells; for example,
to delete genes
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Lambda
• Lambda phage
integration/excision
requires host factor and
two phage proteins
(Int,Xis)
• This means the system
cannot easily used in
other cells
What if you had a way of integrating/excising
DNA that just needed one enzyme and a short
DNA sequence…
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Phage P1
Enterobacteriophage P1
94.8 kb linear, dsDNA
genome
87 x 216 nm
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Infects Shigella, E.coli
Temperate, forms a
plasmid inside cell
Has a site-specific
integrase, Cre. Why?
Dr Maria Schnos, Department of Molecular Virology,
Bock Laboratories, University of Wisconsin
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Cre & LoxP
•
Cre is a 38 kDa, recombinase encoded by P1 phage
•
In the P1 life cycle it causes cyclization of the linear
genome and resolution of multimeric genomes
produced during replication
•
Acts at specific sites (loxP) that are composed of near
identical Cre binding sites (13 nt) in inverted
orientation, with an 8 bp central cross-over region.
•
Simple system: only need Cre and target sequence
(lox)
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Cre - lox recombination
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Cre - lox recombination
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Site-Specific recombination
Reaction intermediate
(from crystal structure of
phage P1 integrase)
showing the close
proximity of the
recombinase enzymes
and the DNA ends.
Strand exchange takes
place in a central cavity of
the protein complex that
contains the central part of
the cross-over region.
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Site-Specific recombination
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Phage P1: Cre-lox
http://www.callutheran.edu/BioDev/omm/jmol/cre/cre.html
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Cre & loxP
•
•
•
•
Cre-lox system will work on ANY DNA in both
eukaryotic and prokaryotic cells.
If you can supply Cre to cell DNA containing lox
sequences, then recombination will occur
Can set this up to show expression from eukaryotic
promoters, eg. Tissue specific gene expression
For example, if put Cre gene under host promoter
control, then give it a target of lox sites that, if excised
will give a signal, then can tell in which tissues the
promoter is active…
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Site-specific recombination
Know the process of sitespecific (specialised)
recombination and be able
to use the lambda and P1
Cre-lox systems as
examples
Be able to compare these to
other DNA transactions that
you learn in this course.
Find an example in the
journal literature where crelox system is used.
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Lambda recombination
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