Bacteriophages: basic characteristics, biology and diversity.

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Bacteriophages basic characteristics, biology
and diversity.

• Dr Mike Dyall-Smith, lab 3.07
• mlds_at_unimelb.edu.au

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What are bacteriophages?

• Viruses of bacteria - first discovered 1915,
  Frederick Twort and but largely researched by
  Felix DHerelle
• Bacteriophages (or phage)
• are small (lt 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
• mlds_at_unimelb.edu.au

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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 recombinationor 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
  site-specific 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 (lt0.1) 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 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 site-specific (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 cre-lox system is used.
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Lambda recombination