Bacteriophage

Titik Nuryastuti
Departemen Mikrobiologi FK UGM

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 • Definisi
• Struktur
• Klasifikasi
• Siklus
• Cara deteksi
• Aplikasi/manfaat

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 Bacteriophage
 Prokaryotes as host
 Subcellular structure without metabolic machinery
 Double stranded DNA, single stranded DNA, RNA
 Virulent phage vs. temperate phage

MS2 T2 Fd, M13

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BC Yang
 Bacteriophage (Phage)
Faga
• Definition - Obligate intracellular parasites that
multiply inside bacteria by making use of some
or all of the host biosynthetic machinery
• genome may be DNA or RNA, single- or double-
stranded, circular or linear, and is generally
present as a single copy.
• Morphology varies from simple, icosahedral and
filamentous phages to more complex tailed
phages with an icosahedral head

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 Historical context
 A century ago, Hankin (1896) reported that the waters of
the Ganges and Jumna rivers in India had marked
antibacterial action (against Vibrio cholerae, restrict
epidemic) which could pass through a very fine porcelain
filter; this activity was destroyed by boiling.
 Edward Twort (1915) and Felix d'Herelle (1917)
independently reported isolating filterable entities capable
of destroying bacterial cultures and of producing small
cleared areas on bacterial lawns.
 It was F d'Herelle, a Canadian working at the Pasteur
Institute in Paris, who gave them the name
"bacteriophages"-- using the suffix phage (1922).

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 Composition and Structure
• Composition
• Nucleic acid Head/Capsid
• Genome size
• Modified bases
• Protein
• Protection
Contractile Tail
• Infection Sheath
• Structure (T4) Tail Fibers
– Size
Base Plate
– Head or capsid
– Tail
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Copyright © 2009 Pearson Education, Inc. Figure 6.14
 Infection processes

1. Attachment of virion to cell

2. Entry of viral nucleic acid into host cell (with
or without other virion components)
3. Early viral proteins synthesized (required
for genome replication)
4. Genome replication
5. Late proteins synthesized (capsid proteins)
6. Assembly of progeny virions
7. Release of infectious progeny virions

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Adsorption and DNA injection

 A random collision,
protein/protein interaction
 Affected by Ca++, Mg++
 Receptor specific (outer
membrane protein lamB for
lambda phage ; sex pili for Qb)

 DNA is the major material

entering bacterial
 Lysozme like activity, core
boring through the cell wall

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BC Yang
Siklus hidup

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 Types of Bacteriophage

• Lytic or virulent phage: Phage that can

only multiply within bacteria and kill the
cell by lysis. (e.g., T4)

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Klasifikasi
• Lytic infection
• infection phage DNA induces switching of the
protein machinery of the host bacterium for
the benefit of infectious agents to produce 50-
200 new phages.
• To make so many new phages requires
nearly all the resources of the cell, which
becomes weak and bursts.
• In other words, lysis takes place, causing
death of the host bacterial cell. As result new
phages are released into the extracellular
space
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 Lytic Life Cycle Overall

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 Kinetics of phage infection
 0 min. Attachment of T2 to a susceptible E. coli cell
 1 min. Inject DNA into cell
 1-7 min. Transcribe and translate early genes
 block bacterial DNA synthesis and degrade host chromosomal DNA
 block transcription of host mRNAs
 block translation of host proteins
 small amounts of early proteins produced (catalytic functions)
 transcription from single phage genome
 7-15 min. Replication of phage DNA
 10-20 min. Translation of phage late proteins (structural)
 transcribed from new phage DNA (many copies of template)
 need large amounts of these proteins to build new virions
 18-25 min. Assembly of new phage particles (end of eclipse
period)
 25 min. Lysis of host cell and release of progeny (end of
latent period)

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BC Yang
 Types of Bacteriophage
• Lysogenic or temperate phage: Phage
that can either multiply via the lytic cycle or
enter a quiescent state in the bacterial cell.
(e.g., )
• Expression of most phage genes repressed
• Prophage
• Lysogen

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Klasifikasi...
• Lysogenic, or temperate
• integration of the phage DNA into the host cell
genome, it may also exist as a plasmid.
Incorporated phage DNA will be replicated
along with the host bacteria genome and new
bacteria will inherit the viral DNA.
• Such transition of viral DNA could take place
through several generations of bacterium
without major metabolic consequences for it.
• certain conditions impeding the bacterium
state, will revert to the lytic cycle

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 Lysis or Lysogeny

• Lysis: Infection by phage produces many

progeny and breaks open (lyses) the host
bacterium
• Lysogeny: After infection, the phage DNA
integrates into the host genome and resides there
passively
– No progeny
– No lysis of the host
– Can subsequently lyse (lysogeny)
• Bacteriophage lambda can do either.
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 UV Induction

Lysis
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Lysogeny
 • Lysogeny occurs when:
• the phage DNA integrates into the bacterial
chromosome
• it is replicated along with the chromosome
• it is passed to daughter cells
• Bacteria containing a prophage are
lysogenic and can grow and divide stably
until viral reproduction is induced.

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 Elements of lysogeny
• The phage genome integrated into the host
bacterial genome is a prophage.
• Bacterium carrying the prophage is a lysogen.
• Lysogens are immune to further infection by
similar phage because the phage functions are
repressed in trans.
• Induction of the lysogen leads to excision of
the prophage, replication of the phage DNA,
and lysis of the host bacterium.

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 Return to be a killer
 Prophage:
 Bacterium is now immune to infection by another phage,
because repressor continuously produced ----- new phage
DNA can be injected into cell and is circularized but is not
transcribed or replicated.
 Prophage can be excised when host give
response to potentially lethal situations:
 if host DNA damaged
 one reaction by host cell is to activate a protease
 protease also cleaves repressor
 Phage DNA now transcibed including a gene for an enzyme
that cuts prophage DNA from bacterial chromosome
 Lytic cycle can start.

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BC Yang
 Lysogenic cycle
Lysogenic Cycle: Lambda as an example

 lambda integrase and lambda repressor cI

synthesized due to activation of the
transcription of their genes by cII.
 cI repressor turns off phage transcription
 integrase catalyzes integration of lambda
DNA into bacterial chromosome via short
sites of homology (site-specific
recombination) ---- prophage

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BC Yang
Lytic vs Lysogenic Cycle?
Rely on environmental conditions :
UV, chemical agent, etc.

• Role of repressor (CI encodes the

repressor that turns off lytic functions)
• Role of cro gene product (encodes the
"antirepressor" that turns off the repressor)
• Role of proteases

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 Bakteriofaga

CARA ISOLASI

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 Assay for Lytic Phage
Phage

• Plaque assay Method

Bacteria
– Plaque forming unit +
(pfu) Phage

– Measures infectious
particles

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 Glossary
 pfu: plaque forming unit
 Title: define pfu in a phage suspension
 moi: multiplicity of infection, the ration of
phage particles to bacteria
 eop: efficiency of plating, the ration of the
plaque titer to the number of phage particles
 Prophage: state of phage co-existing with host
 Lysogenic bacteria: term of bacteria carrying
prophage
 Phage conversion: phenotype change in
lysogenic bacteria

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BC Yang
Phage isolation

• ..\Exercise37 Faga isolation.pdf

• Faga E. coli

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 plaque
 Plaques are clear zones formed
in a lawn of cells due to lysis by
phage.
 At a low multiplicity of infection
(MOI) a cell is infected with a
single phage and lysed,
releasing progeny phage which
can diffuse to neighboring cells
and infect them, lysing these
cells then infecting the
neighboring cells and lysing
them, etc,
 It results in a circular area of
cell lysis in a turbid lawn of
cells.

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BC Yang
 Isolation of Bacteriophage

Samples with Enrichment

broth (sewage, river...)

Incubation

Shaking and Centrifugation

Chloroform Precipitation,
Centrifugation and
Filtration

Mass Production

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 Temperate and lytic phage have a different
plaque morphology
T emperate phage Mutants of phage that
generate turbid have lost the capacity to
plaques lysogenize form clear
plaques

lysogenized cells

lysed cells lysed cells

uninfected cells

Lytic phage: clear plaques

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 Mass Culture- Bacteriophage Suspension

Sewage/ Sludge
(Broth Culture)
Incubation

Shaking Incubation

Centrifugation
Rinsate Centrifuge
Shaker incubator

Chloroform – Enriched single type of

phage

Lyophilised

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Manfaat

 Phage typing : Identification of bacteria -

phage typing
 Model system of molecular biology
 Medical applications : phage as natural, self-
replicating, self-limiting antibiotics.
• Treatment and prophylaxsis???
 Gene transfer in bacteria
 Sebagai kendaraan untuk memindahkan gen
dengan proses transduksi.

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 Medical Applications of
Phage
• Exponential Biotherapies (Rockville, MD)
• Vancomycin resistant Enterococcus facium and
Streptococcus pneumoniae
• Phage Therapeutics (Bothell, WA)
• Staphylococcus aureus and Staphylococcus epidermidis
• Intralytix, Inc. (Baltimore, MD)
• Salmonella in meat and poultry
• Biopharm Ltd. (Tblisi, Georgia)
• Infections associated with burns
• University of Idaho
• Escherichia coli O157:H7 in cattle
Reassessment of Medicinal Phage Spurs Companies to Study Therapeutic Uses. American
Society for Microbiology News 64:620-623, 1998.
Phages eyed as agents to protect against harmful E. coli. American Society for
Microbiology News
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Inc. 1999.
Significance of Lysogeny

• Model for animal virus transformation

• Lysogenic or phage conversion
• Definition: A change in the phenotype of a
bacterial cell as a consequence of lysogeny
• Modification of Salmonella O antigen
• Toxin production by Corynebacterium diphtheriae

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 PHAGE THERAPY

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 OTHER APPLICATION

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 Application of Bacteriophages

Application of Mass culture with pre-determined doses for

removal of E.coli and other fecal coliform;

 Sewage Treatment

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 Terima Kasih

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 Life Cycle of Bacteriophage
Lytic cycle:
a) Phage Adsorption on Host Cell.
b) Genetic Material Penetration of Phage.
c) Phage Multiplication.

d) Complete assemblage of Phages.

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 Lysogenic cycle:
The genetic material of Phage remains within the host cells and
reproduce in synchrony with the host for long-period.
Lysogenic and Lytic cycle

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Copyright © 2009 Pearson Education, Inc. Figure 6.18
Section 6.7

• In generalized transduction, bacterial

DNA instead of phage DNA is packaged in
a phage particle and transferred to a
recipient host (Figure 6.18).
• In specialized transduction, a small
piece of bacterial DNA is packaged along
with the phage DNA.

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Section 6.8

• 6.8Bacteriophages Undergo Intergenic

Recombination

• Phage mutations often affect plaque

morphology (Figure 6.19 and Table 6.1).
• Such mutations have been important in
understanding genetic phenomena in
phages.

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Section 6.8

• Mapping in
Bacteriophages
• Mixed infection
experiments
demonstrated that
intergenic
recombination
occurs in
bacteriophages.

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 Induction and immunity of lysogens


A  lysogen
Spontaneously,
1/1000 lysogens will +
induce, i.e. the l UV treatment leads
prophage will Lysogens are immune to
to induction of further infection with similar
excise, replicate and virtually all lysogens
lyse the cell. (lambdoid) phage
in a culture.

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One step growth
demonstrate an eclipse period during which the DNA began
replicating and there were no free phage in the cell, a period
of accumulation of intracellular phage, and a lysis process
which released the phage to go in search of new hosts.

Ellis, E. L. and M. Delbrück (1939). The Growth of

Bacteriophage. J. Gen. Physiol. 22:365-384.
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Life cycle
of phage 

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 Events Leading to Lysogeny
• Circularization of the phage
chromosome
• Cohesive ends
Cohesive Ends

Lygase

Linear Double Stranded Opened Circle Closed Circle

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 Lytic Phage Multiplication Cycle

• Eclipse

Number of Infectious Particles

Total Extracellular
Phage Phage
• Early genes
• Phage DNA
synthesis
• Late genes
• Intracellular
Intracellular
Lysis
accumulation Eclipse accumulation
phase
• Lysis and Release
Time after Infection

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Lytic cycle of phage

5 6 7
3
1 4

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BC Yang