1. Self assembly and Applications
Bacteriophages
Self assembly and Applications

2. Bacteriophages: Definition & History
Bacteriophages are viruses that can infect and destroy bacteria.
They have been referred to as bacterial parasites, with each phage type depending on a single strain of bacteria to act as host.

3. Bacteriophages: Classification
Based on two major criteria:
phage morphology (electron microscopy)
nucleic acid properties

4. Bacteriophages: Classification
At present, over 5000 bacteriophages have been studied by electron microscopy and can be divided into 13 virus families.

5. BACTERIAL CELL INFECTION BY VIRUS
BACTERIAL CELL INFECTION BY VIRUS * Virus binds to receptor and ejects genome *Viral particle stays outside cell! Only its genome enters *Virion leaves via lysis of cell

6. 13 Bacteriophage families
Double stranded DNA,
Non-enveloped
Double stranded
DNA, Enveloped
SIRV 1, 2 P2
Rudiviridae
Myoviridae T2
Plasmaviridae
Fuselloviridae
SSV1
TTV1 λ
Tectiviridae
PRD1
Siphoviridae
Lipothrixviridae
PM2
P22
Corticoviridae
Podoviridae
Single stranded
RNA
Double stranded RNA
Single-stranded DNA
M13 & fd
Inoviridae
MS2
phi666
ΦX174
Leviviridae
Microviridae
Cystoviridae

7. 13 Bacteriophage families
Corticoviridae
icosahedral capsid with lipid layer, circular supercoiled dsDNA
Cystoviridae
enveloped, icosahedral capsid, lipids, three molecules of linear dsRNA
Fuselloviridae
pleomorphic, envelope, lipids, no capsid, circular supercoiled dsDNA
Inoviridae genus
(Inovirus/Plectrovirus)
long filaments/short rods with helical symmetry, circular ssDNA
Leviviridae
quasi-icosahedral capsid, one molecule of linear ssRNA
Lipothrixviridae
enveloped filaments, lipids, linear dsDNA
Microviridae
icosahedral capsid, circular ssDNA
Myoviridae (A-1,2,3)
tail contractile, head isometric
Plasmaviridae
Podoviridae (C-1,2,3)
tail short and noncontractile, head isometric
Rudiviridae
helical rods, linear dsDNA
Siphoviridae (B-1,2,3)
tail long and noncontractile, head isometric
Tectiviridae
icosahedral capsid with, linear dsDNA, "tail" produced for DNA injection

8. Bacteriophages: Virulence Factors Carried On Phage
Temperate phage can go through one of two life cycles upon entering a host cell.
Lytic:
Is when growth results in lysis of the host and release of progeny phage.
Lysogenic:
Is when growth results in integration of the phage DNA into the host chromosome or stable replication as a plasmid.
Most of the gene products of the lysogenic phage remains dormant until it is induced to enter the lytic cycle.

9. Bacteriophages: Lysogenic Conversion
Some lysogenic phage carry genes that can enhance the virulence of the bacterial host.
For example, some phage carry genes that encode toxins.
These genes, once integrated into the bacterial chromosome, can cause the once harmless bacteria to release potent toxins that can cause disease.

10. Bacteriophages: Lysogenic Conversion
Examples of Virulence Factors Carried by Phage
Bacterium
Phage
Gene Product
Phenotype
Vibrio cholerae
CTX phage
cholerae toxin
cholera
Escherichia coli
lambda phage
shigalike toxin
hemorrhagic diarrhea
Clostridium botulinum
clostridial phages
botulinum toxin
botulism (food poisoning)
Corynebacterium diphtheriae
corynephage beta
diphtheria toxin
diphtheria
Streptococcus pyogenes
T12
erythrogenic toxins
scarlet fever

11. Bacteriophages

12. Bacteriophages: HK97 assembly

13. Bacteriophages The filamentous Phage f: A: AFM image
B: Schematic representation
1: initiation of assembly
2,3: elongation
4: termination

15. Bacteriophages

17. Bacteriophages Used for cloning foreign genes among other applications
Proteins and peptides are fused to the Capsid(surface) of the phage
The combination of the phage and peptide is known as a Fusion Protein

18. Bacteriophages
Different sets of genes are inserted into the genomes of multiple phages
These separate phages will only display one protein, peptide, or antibody
Collections of these phages can comprise Libraries
These Libraries are exposed to selected targets and only some phages will interact with targets

19. Bacteriophages
3 types of common phages used in phage display are the M13, F1 , FD
Virions take up a small amount of area
Through using multiple Virions polypeptide libraries can be created, and each phage displays a random peptide

20. Bacteriophages

21. Bacteriophages

22. Bacteriophages

23. Bacteriophages
By taking gene segment of antigens of antibodies and fusing them to the protein coat of phages, these phages will now express the anti-body in a fusion protein
Phage Display Libraries of antigens can be created to create anti-body phage display libraries

24. Bacteriophages
Polypeptides of interest can be screened using selection techniques
The target protein/peptide can be immobilized using magnetic beads
With the advance of DNA sequence recognition these selected sequences can be identified easily

25. Bacteriophages

26. Bacteriophages
Phage display

27. Bacteriophages
Once these Phages are isolated and recovered they can be used to infect bacteria which will create a particle similar to a monoclonal antibody

28. Bacteriophages
A: wt; B-F: types of pIII displays; G: pVII or pIX display H: mosaic pVIII display, I:uniform pVIII display

29. Bacteriophages Morphology of the T series of Phages Name Plaque size
Head (nm)
Tail (nm)
Latent period (min)
Burst size T1
medium 50
150 x 15 13
180 T2
small
65 x 80
120 x 20 21
120 T3
large 45
invisible
300 T4
23.5 T5
100
tiny 40 T6
25.5 T7

30. Bacteriophages

31. Bacteriophages

33. Bacteriophages

34. Bacteriophages
The Phages HK97 and HK022 do have a very prominent friend the bacteriophage λ

35. Properties of Filamentous Viruses
fd Pf1 Pf3 PH75
Symmetry class I (C5S2)a II (C1S5.4)b II (C1S5.4)b II (C1S5.4)c Length (nm) Ext. diam. (nm)
No. subunitsd No. nucleotides Nucl./subunit Wt-% protein
a10 subunits per 32 Å helical repeat. Marvin et al. (2006) J. Mol. Biol. 355,
b27 subunits per 75 Å helical repeat. Welsh et al. (2000) Acta Cryst. D56, ; Welsh et al. (1998) J. Mol. Biol. 283,
cPederson et al. (2001) J. Mol. Biol. 309,
dSequences: fd: AEGDDPAKAA FDSLQASATE YIGYAWAMVV VIVGATIGIK LFKKFTSKAS50 (5.24 kDa; pI = 6.3)
Pf1: GVIDTSAVES AITDGQGDMK AIGGYIVGAL VILAVAGLIY SMLRKA46 (4.61 kDa; pI = 4.7)
Pf3: MQSVITDVTG QLTAVQADIT TIGGAIIVLA AVVLGIRWIK AQFF44 (4.63 kDa; pI = 5.7)
PH75: MDFNPSEVAS QVTNYIQAIA AAGVGVLALA IGLSAAWKYA KRFLKG46 (4.81 kDa; pI = 9.4)
Note that Pf3 is similar to Ff in composition, but more similar to Pf1 in assembly architecture. Pf3 subunit has one Trp (W38), as does Ff subunit (Trp 26).
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36. fd Architecture Filament of 6.5 x 880 nm (PL = 2 μm)
1/100th virion length
ssDNA
core
65 Å
Filament of 6.5 x 880 nm (PL = 2 μm)
Coat of layered -helical subunits
Arranged with 5-fold rotational symmetry
Right-hand slew on capsid surface
ssDNA packaged within (conformation?)
Caspar & Makowski (1981) J. Mol. Biol. 145,
Day et al. (1988) Ann. Rev. Biophys. 17,
Marvin et al. (1994) J. Mol. Biol. 235,
fd (6.5 x 880 nm)
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37. Molecular Models of the fd Capsid
Zeri et al. (2003) PNAS 100,
solid state NMR
Marvin et al. (2006) J. Mol. Biol. 355,
fiber X-ray diffraction
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38. Bacteriophages