Bacteriophages

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Bacteriophages

• The Importance of Bacteriophages

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Historical

• Frederick Twort (1915) and Felix d'Herelle (1917)
  were the first to recognize viruses which infect
  bacteria, which d'Herelle called bacteriophages
  (eaters of bacteria). In the 1930s and subsequent
  decades, pioneering virologists such as Luria,
  Delbruck and many others utilized these viruses
  as model systems to investigate many aspects of
  virology, including virus structure, genetics,
  replication, etc. These relatively simple agents
  have since been very important in the development
  of our understanding of all types of viruses,
  including those of man which are much more
  difficult to propagate and study. They are still
  a paradigm for many areas of biology, especially
  gene expression.

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Environmental

• Bacteriophages, like bacteria, are very common in
  all natural environments and are directly related
  to the numbers of bacteria present. They are thus
  very common in soil and have shaped the evolution
  of bacteria.

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Industrial/Economic

• Phages of Lactobacillus are a serious problem for
  the dairy industry.Medical - phage typing (e.g.
  Staphylococcus) antibacterials -
  Flemming.Recombinant DNA vectors - cloning,
  expression, enzymes (T4 DNA ligase)

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Diversity

• There are at least 12 distinct groups of
  bacteriophages, which are very diverse
  structurally and genetically the best known ones
  are the common phages of E.coli

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Replication
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The Single Burst Experiment (Ellis and Delbruck,
1939)
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The Single Burst Experiment (Ellis and Delbruck,
1939)
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The Hershey-Chase Experiment (1952)
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Bacteriophage T4

• Family Myoviridae
• Order Caudovirales

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What is bacteriophageT4?

• DNA is packaged in the head of T4 phage.
• The tail shaft is composed of two concentric
  cylinders. The outer cylinder is called tail
  sheath, and it will contract upon infection.
• The hexagonal baseplate is made of 22 kinds of
  proteins.
• The tail fibers are responsible for recognizing
  the receptor in the outer membrane of the host
  cell.

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Why is bacteriophage T4 a good research material?

• totally determined nucleotide sequence
• various mutants available
• observable structural transformations by electron
  microscopy
• various intriguing structures such as spherical
  structures, cylinder structures, fibrous
  structures, and so forth
• available in vitro synthesis.

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Morphology

• Virions not enveloped tailed head. Head
  separated from tail by a neck, tail complex,
  consisting of a central tube and a contractile
  sheath, provided with a collar, base plate, 6
  short spikes and 6 long fibers. Nucleocapsids
  isometric to quasi-isometric elongated 65-115 nm
  in diameter (95-)111 nm long (65-)80 nm in
  diameter. Symmetry icosahedral. Nucleocapsids
  appear to be angular. 152 capsomers per
  nucleocapsid. Tail contractile (80-)113(-455) nm
  long 16 nm wide.

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when the bacteriophage T4 is assembled

• no other helps are needed.
• the proteins are interacting themselves by
  changing conformation.
• No external energy is needed.

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Nucleic Acid

• Virions contain 48 nucleic acid. Virions
  contain one molecule of linear double stranded
  DNA.
• Total genome length is 336000 nt. Double stranded
  DNA circularly permuted. Genome sequence has
  terminal repeated sequences. Guanine cytosine
  ratio 35 . Special nucleotides found in genome,
  are 5-hydroxy-methyl cytosine (instead of
  thymidine).

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Structural protein

• PDB Id1ocy
• NameStructural protein
• TitleStructure of the receptor-binding domain of
  the bacteriophage t4 short tail fibre
• StructureBacteriophage t4 short tail fibre.
  Chain a. Fragment receptor-binding domain,
  residues 330-527. Engineered yes
• Source Bacteriophage t4. Strain d. Expressed
  in escherichia coli. Expression_system_variant
  de3. Other_details variant as present in
  laboratory of s. Miller. Co-expression with gp57
  chaperone

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Main view Bottom view
Right view
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Virulent vs. Temperate Phages

• Virulent phages do not integrate their genetic
  material into the host cell chromosome and
  usually kill the host cells (lytic infection)
  (e.g. T-phages of E.coli).
• Temperate phages may integrate into the host DNA,
  causing LYSOGENY.

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Virulent Bacteriophages

• The archetypal virulent bacteriophage is T4 which
  carries a genome of 173 kb of linear ds DNA.
  During the early stages of a infection cycle T4
  nucleases encoded by socalled early genes degrade
  the chromosomal DNA of E. coli in order to obtain
  large quantities of nucleotide precursors for its
  own DNA syntesis.

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Virulent Bacteriophages

• For this mechanism to work T4 had to distinguish
  its own DNA from the host cell's by incorporating
  a modified base, 5-hydroxymethylcytosin which
  replaces the normal cytosin. In addition T4 also
  had to evolve genes and enzymes which reduce the
  large pool of dCTPs accumulating during the
  degradation of E. coli DNA. This is achieved by
  two other phage encoded enzymes called dCTPase
  and dCDPase generating dCMP which is unavailable
  for DNA polymerization.

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Virulent Bacteriophages

• E. coli however has evolved a defence mechanism
  against bacteriophage T4 infection by producing
  endonucleases encoded by a gene called rglA
  these enzymes are directed against DNA containg
  5-hydroxymethylcytosin. Bacteriophage T4 has
  responded to this challenge by glycosylating the
  above base via a glycolylase rendering the host's
  endonucleases useless.

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Lyctic cycle of bacteriophage T4
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Lyctic cycle of bacteriophage T4

• The lytic cycle of the infection of E. coli
  starts with the adsorption of T4 to specific cell
  surface receptors which often consist of LPS
  Then the tail section contracts thus penetrating
  the relatively regid peptidoglycan cell wall of
  the bacterial cell. The DNA residing in the phage
  head is then injected into the interior of the
  bacterial cell. From then on a series of
  molecular events occur which lead to the
  formation of phage capsid (head and tail)
  proteins which assemble (together with the phage
  DNA) in a process known as maturation to form
  fully infectious phage particles.

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Virulent Bacteriophages

• The infection E. coli by bacteriophage T4 is a
  typical example of a host-parasite evolutionary
  race in which one partner tries to outsmart the
  other one by generating novel functions.

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Virulent Bacteriophages

• One final note Bacteriophage T4 does not encode
  its own RNA polymerase instead it successively
  modifies the existing E. coli RNA polymerase and
  in doing so alters its promoter specifities first
  to recognize the T4 promoters for the early genes
  (see above) and later in the infection cycle to
  transcribe promoters for the late genes encoding
  head and tail protein.

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Lyctic cycle of bacteriophage T4

• One of the phage proteins produced is lysozyme an
  enzyme which degrades peptidoglycan. This leads
  to a disintegration of the cell wall accompanied
  by a release of mature phage particles. The
  number of phage particles produced per infection
  cycle is known as the burst size. The diagram
  above gives an illustration of a lytic infection
  cycle but note that for T4-mediated infections
  the E. coli DNA (in the diagram as red circle) is
  actually degraded.

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The Single Burst Experiment (Ellis and Delbruck,
1939)
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The Single Burst Experiment (Ellis and Delbruck,
1939)
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Lyctic cycle of bacteriophage T4
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Phage Genetics

• TRANSDUCTION. There are two forms Generalized
  Transduction bacterial rather than phage DNA is
  packaged into a phage head. When another cell is
  infected, the bacterial DNA is injected and in a
  proportion of cases, may be incorporated into the
  chromosome by homologous recombination, replacing
  the existing genes. Frequency 105 - 108 per cell.
  More than one gene may be cotransduced - limit
  packaging size 50kbp 1 of bacterial
  chromosome.

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Phage Genetics

• Specialized Transduction Results from inaccurate
  excision of an integrated prophage some phage
  DNA is lost and some bacterial genes are picked
  up and carried to the next host - therefore phage
  are usually defective (non-infectious) and
  require replication-competent helper phage to
  replicate, depending on which phage genes are
  lost.

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Bacteriphage infection can be easily monitored by
placque formation. (in the lytic cycle, anyway)
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THE END