3. Bacteriophage(ubiquitous)
HISTORY
1896 Ernest Hanburg Hankin –antibacterial action against cholera and
it could pass a very fine porcelain filter.
1898, Gamalega –observed similar phenomenon in bacillus subtilis
1984-samsygina and boni –reported such phenomeon.
1915- ferederick william Twort-presence of a virus
D’Herelle (1917) coined the term bacteriophage meaning” bacteria
eater” (observed that filtrate from feces culture from dysentry)
The greek word backerion means “bacteria” and phagein means “to eat”
He suggested that lytic agent was a virus and named
bacteriophage(twort-d herelle phenomenon)
Jules bordet and andre grata –rediscovered it
Salvador e.luria –get clear image of a bacteriophage using electron
microscope
4. history
1940-delbruck - worked out the lytic mechanism
Luria ,delbruck and hershey-studied genetic changes occur
when viruses infect bacteria
1952-proved DNA That transmits the genetic information
Shared their nobel prize for physiology in 1969
1952-norton zinder and joshua loderberg-phage can
incorporate its genes into the bacterial chromosome
1980-frederick sanger –awarded nobel prize for his
discovery of determing the nucleotide sequence in dna
using bacteriophages.
5. BACTERIOPHAGES
Introduction
A virus that infects
Replicates within a
bacterium
Derived from”bacteria”
and phagein to “devour”
Single stranded or
double stranded
Surrounded by protein
capsid(subunits
capsomere)
Genomes may encode
as few as four genes and
as many hundreds of
genes.
6. Bacteriophage
Unenveloped ,complex symnetry
Up to 70% of marine bacteria may be infected by
phages
19 families are currently recognized by the ICTV that
infect bacteria and archea.
9families infect bacteria only
9 infect archea only, and one (tectiviridae) infects both
bacteria and archea.
Two life cycles ,lytic (virulent)or lysogenic (temperate)
7.
8. classification
The ICTV classifies the bacteriophage under an order
Caudovirales according to morphology and nucleic
acid.
On the basis of presence of single or double strands of
genetic material the bacteriophage are the following
types.
SS DNA bacteriophage(fd,fi,m13)
Ds DNA bacteriophaget2,t4,t6,t1,t3,t5,t7etc)
Ss RNA bacteriophage(m12,ms2,r17)
Ds RNA bacteriophage
9. Bacterial viruses may grouped into
6 morphological types
TYPE A:this most complex type has a hexagonal head,a
rigid tail with a contractile sheath and tail fibres.
TYPE B:similar to A, this type has a hexagonal head,a rigid
tail with a contractile sheath its tail is flexible,and it may or
may not have tail fibres.
TYPE C:this type is characterised by a hexagonal head and
a tail shorter than the head .the tail has no contractile
sheath and may or may not have tail fibres
TYPE D: this type has a head madeup of large
capsomeres,but has no tail
TYPE E: this type has a head madeup of small capsomeres
but has no tail
TYPE F: this type is filamentous
12. MORPHOLOGY
Tadpole shaped,with
hexagonal head and
cylindrical tail.
Contains 5 important
substructures
(head,head-
tailconnector,tail base
plateand fibres)
Heads consists of a
tightly packed core of ds
DNA surrounded by a
protein coat or
capsid.(elongated,bi-
pyramidal)
Size 95*65 contains
2000 identical protein
subunits(capsomeres)
13. MORPHOLOGY
The tail composed of a contractile sheath
surrounding the hollow core and contains 24
annular rings (each containing 6
subunits)constitute the sheath
It connected to the head with a connector
having a collar with attached whiskers
Size of the tail is 80*18nm
Terminal base plate having prongs or tail fibres
attached
The base plate -6 spikes as its six corners
Former-helpd in recognition of specific
receptor sites
dsDNA -50 (mu m)
DNA-1000 longer than phage
Circular and terminally redundant
T-even phages -5- hydroxymethylcytosine
instead of cytosine
Synthesis of phage occur easily.
14. Diseases caused by
bacteriophages
Bacteriophages only infect
bacteria,they do not cause disease
in humans.
Alter the genome on non- virulent
bacterial strains; thus producing
more virulent strains examples are:
CHOLERA
Most strains of cholera are
harmless.
Responsible for producing harmful
cholera strains.
15. Bacteriophages
SCARLET FEVER
Commonly affects children
Signs and symptoms include sore throat, fever and a
characteristic red rash.
Usually spread by inhalation
There is no vaccine of this disease.
Most of the clinical features are caused by
erythrogenic toxin, a substance produced by the
bacterium streptococcus when it is infected by
bacteriophage T12
16. Higher classification: Tequatrovirus
Scientific name : Escherichiavirus t4
Realm : Duplodnaviria
Family : Mycoviridae
Order : Caudovirales
17. T4 bacteriophage
Ubiquitously distributed in nature
Ranging from mammalian gut to soil ,sewage and oceans
130 similar morphological features as phaget4 has been
described
App 1400 major capsid protein sequence have been
correlated with its 3D structure
Large enlongated head,contractile tail and a complex
baseplate with 6LTF
It serves as an excellent model for large icosahedral viruses
Herpes virus
It was first observed by negative stain electron microscopy
18.
19. Head structure(head proteins)
194 MDa mature head encapsidates
Empty capsid- ATP-dependent packaging machine.
Prolate head-Procapsid,capsid,polyhead
Hoc(highly antigenic outer capsid protein) and soc (small outer capsid
protein)
Soc (Mr=10,000) present in the Capsid equivalent to the number of p23
molecules
Hoc(mr= 40,000)is lower than that of p23 molecules
These proteins are distributed over the surface of the heads of the antibody
bound capsid
These proteins basically not changed the cryo-electron microscopic structure
determination of isometric capsids
Dimension of the phage altered the cryo electron microscopy
Prolate icosahedron are Tend=13 laevo and tmid=20(86nm wide and 120nm
long)
Capsid is composed of 930 post translationally modified monomer
20. Cont……
Or 155 hexamers of the major protein
gene product 23(gp23*)
Gp24* forms pentamers at 11 of the
12 pentameric vertices and is hologus
to gp23* with 21% residues
12th vertex –special vertex (portal
protein gp20)
Portal protein- initiates head
assembly,genome packing ,genome
gatekeeper)
Rod shaped soc binds between 2
gp23*hexamer
Soc maintains the stability of the
head
Hoc is an elongated molecule
protruding from center of gp23*
hexamers
21. `
the major Capsid protein gp23*
and the vertex protein gp24*
similar in mw and sequence
•Hk97-like capsid protein fold
•17% of the structurally aligned
residues between t4gp24 and
hk97 were identical
Structyrally aligned residues
(axial and peripheral domain)
A domain forms central part of
hexameric and pentameric
capsomerrs
P domain located in
capsomeres periphery
Several stages of head prohead
formation,prohead
proteolysis,dna
pacaking,expansion of the
porlated head and binding of spc
and hoc ;
22. Initiation complex –portal
protein gp20 mediated by
chaperone protein gp40
Prohead core proteins
gp21,gp22,gp67,gp68
Initiation proteins
IPI,IPII,IPIIIV and gpalt
Gp23 and gp24 start to form a
shell around the core protein
To assemble prohead
Gp23 requires 2 chaperone
protein –GroEL,gp31
Assembly of prohead -930 copies
of gp23 and 55 copies of gp24
Volume of prohead is app 15-20%
smaller than mature head
Gp21-protease (t4PPase) in
prohead
Cleaved head is released from
membrane into cytosoland
gp23*and gp24* leads to larfe
conformational expansion of the
prohead.
23. gp17 and gp16 complex cleaves
the dna and associates the
cleaved end
Terminase –DNA complex
created
Dodecameric portal protein
gp20 at the vertex of the
prohead
Gp16 initiates the dna
translocation by ATPase
activity of gp17
Capsid expands during the
DNA pacaking
Expansion causes rotation of
the gp23*subunits
50%increase of the capsid
volume and stabilises capsid
and creates binding sites for
soc and hoc
24. Cont..
Motor generates forces app 60pn and trabslocation rates up
to app 2000bp/s
172 kb t4 genome pacakged in app 5 min in concentric
layers’head is filled by DNA , a signal
Internal pressure produced by the pacakaged DNA is
sensed by portal protein and transmitted to gp17
It then cleaves DNA and pentameric gp17 motor is released
from capsid
Mature head is completed by attachment of neck proteins
gp13,14,2 and gp4 to portal vertex
Head is ready to be joined to tail
LTFs produce a mature phage
25. TAIL STRUCTURE
Tail consists of tail tube surrounded by helical sheath
And attachedd to dome shaped baseplate at end from
the head
6STFs folded beneath the baseplate
Assemblies of tail begins with assemblies of 6 wedge
and central hub
Form the baseplate and nucleats tail tube around
which the sheath assembles
Sucrose density graddient centrifugation assay
26. Wedge protein
gp11,gp10,gp7,gp8,gp6,gp53,gp25 are
assembled to form a wegde
6 wegdes associated around the
central hub form a hexagonal
baseplate
Is completed by attachment of STF
sand gp9 at its periphery
Gp48 and gp54 to top of the hub
Creating platform for polymerization
of tail tube (gp19)
And tail sheath (gp18)
Gp29 controls the length of the tail.
Gp3 binds to last row of gp19 thus
stabilizing the polymerized tail tube
Tail sheath polymerises around the
tail tube in parallel with the
formation of the tail tube
Is completed by the binding of tail
terminator gp15 togp3 and the last
row of gp18.
27. Structure and
basseplate
Baseplate undergoes a dramatic
conformational change
Relaxes high energy dome
structure to low energy star shaped
structure
Analyzing the structure of the
component proteins
Crystal structure of nine
baseplate
proteins(gp11,gp10,gp8,gp6,gp25,gp
9,gp12,gp5,gp27)
Structure fitted into 3D
cryoEMreconstructed baseplate
for both dome-shaped and star
shaped conformations
Conformation of the baseplate is
changing from the dome-shaped to
star shaped during infection
process
28. Proteins at the periphery of baseplate
(gp11,gp10,gp9)have 3fold symmetry
Connect the LTFs to baseplate and
transmit signal to baseplate on recognize
a host cell surface lipopolysaccharide
molecule
Attachment of LTFs is gp9 which ia an
elongated trimer has 3 domains
N- terminal domain forms alpha
helical ,triple coiled coil
Middle domain consists of 7 stranded
beta sandwich
C terminal domain -8 stranded
antiparallel beta barrel structure
It assocciate with a trimeric domain
associates with gp7 in the baseplate
Hinge between n-terminal and the
middle domain provides flexibility to
LTFs
Change in gp9 initiates the baseplate
conformational change result in transfer
of t4 genome into the host
Gp10 is the largest protein component
29. Tail tube and sheath
terminators
Polymerized tail tube and sheath
capped by terminator protein gp3
and gp15
Prevent depolymerization before
the tail attaches to the head
Form hexameric rings interact
with last row of gp19 and gp18
molecules
Gp3 has not determined
Gp15 has 4 stranded antiparallel
beta sheet and alpha helix facing
inner side of the ring
Central pore and side surface of
gp15 are negatively charged
Top and bottom surfaces are
positively charged and it interacts
with gp14 and gp3
Interaction between gp15 and gp18
different in extended and
contracted conformations
30. In contacted tail ,-vely charged side surface of the gp15
hexamer interacts with +vely charged surfaces of c-
terminal domain of gp18 molecules
Interactions help to maintain the integrity of the tail
in its contracted form
Gp15 hexamer undergo conformational change during
infection
Propagated through gp14 and gp13 to portal assembly
to allow the release of the genomic DNA
31. LTF COLLAR AND
WHISKERS
1450 A long LTFs consist 4 proteins
Gp34,gp35,gp36 and gp37
Chaperon protein gp57A is
required for trimerization of gp34
,gp37
Chaperon protein gp38 required
proper folding of gp37
Gp34 formed by proximal half of
the fibre and interact with adaptor
protein gp9 on baseplate
Monomeric gp35 forms the hinge
Proximal and distal half fibres
assemble independently
C-terminal part of gp36 binds to n-
terminal region of gp37
Distal part of the fibre divided into
11 domains (D1-D11)
32. Cont…
Domain 1 and domain2 are a part of gp36
D3-D11 are part of gp37
D10 and D11 of gp37 (was in terms of collar ,needle and
head domain)
Head domain sits at tip of the distal end and
recognizes the receptor binding site on the host cell
Gp37 known to bind to lipopolysacharide and as
protein saccharide interactions
Involve aromatic side chains –Trp and Tyr residues at
the tip of gp37 important for host recognition
33. Filled head is attached to the tail via
the neck proteins gp13 and gp14
Six 500 A – long trimeric ‘whiskers’
fibres (gpWac) are atached to the
neck
Whiskers have a trimeric coiled coil
structure known as ‘fibritin’
50 amino acid residues at the n-
terminus of gpWac bind to neck
region
30 amino acid residues 0f the c-
terminus are a chaperon determines
the proper trimerization of the
fibritin
6 other fibres also constructed of
fibritin form a collar around the neck
Fibritin and gp63 (RNA ligase)
promote attachment of the LTFs to
the phage
It is also important in sensing the
optimum environment for phage
infection
34. Complementation assays,cross linking analyses,x-ray
crystallography and cryoEM,providing a structural
model of phage t4