Bacteriophages

Self assembly and Applications

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.

Bacteriophages: Classification

• Based on two major criteria:

phage morphology (electron microscopy)

nucleic acid properties

Bacteriophages: Classification

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

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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

Double stranded DNA, Enveloped

Double stranded DNA,Non-enveloped

Myoviridae

Siphoviridae

Podoviridae

P2

T2

λ

P22

Tectiviridae PRD1

Corticoviridae PM2

Single-stranded DNA

Inoviridae M13 & fd

Microviridae ΦX174 Leviviridae

Single strande

d RNAMS2

Lipothrixviridae

TTV1

Fuselloviridae SSV1Plasmaviridae

Double stranded RNA

phi666

Cystoviridae

RudiviridaeSIRV 1, 2

13 Bacteriophage families

13 Bacteriophage familiesCorticoviridae

icosahedral capsid with lipid layer, circular supercoiled dsDNA

Cystoviridaeenveloped, icosahedral capsid, lipids, three molecules of

linear dsRNA

Fuselloviridaepleomorphic, 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

Plasmaviridaepleomorphic, envelope, lipids, no capsid, circular

supercoiled dsDNA

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

Tectiviridaeicosahedral capsid with, linear dsDNA, "tail" produced for

DNA injection

Bacteriophages: Virulence Factors Carried On Phage

• Temperate phage can go through one of two life cycles upon entering a host cell.

1) Lytic:Is when growth results in lysis of the host and release of progeny phage.

2) 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.

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.

Bacteriophages: Lysogenic Conversion

Examples of Virulence Factors Carried by Phage

Bacterium PhageGene

ProductPhenotype

Vibrio cholerae CTX phage cholerae toxin cholera

Escherichia colilambda phage

shigalike toxinhemorrhagic

diarrhea

Clostridium botulinumclostridial phages

botulinum toxin

botulism (food poisoning)

Corynebacterium diphtheriae

corynephage beta

diphtheria toxin

diphtheria

Streptococcus pyogenes

T12erythrogenic

toxinsscarlet fever

Bacteriophages

Bacteriophages: HK97 assembly

Bacteriophages

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

Bacteriophages

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

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

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

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• 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

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

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Bacteriophages

• Phage display

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

Bacteriophages

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

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 13 300

T4 small 65 x 80 120 x 20 23.5 300

T5 small 100 tiny 40 300

T6 small 65 x 80 120 x 20 25.5 200-300

T7 large 45 invisible 13 300

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

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) 880 1900 680 910

Ext. diam. (nm) 6.5 6.5 6.5 6.5

No. subunitsd 2750 7400 2500 2700

No. nucleotides 6408 7420 5800 6500

Nucl./subunit 2.4 1.0 2.4 2.4

Wt-% protein 87 94 86 87

a10 subunits per 32 Å helical repeat. Marvin et al. (2006) J. Mol. Biol. 355, 294-309. b27 subunits per 75 Å helical repeat. Welsh et al. (2000) Acta Cryst. D56, 137-150; Welsh et al. (1998) J. Mol. Biol. 283, 155-177. cPederson et al. (2001) J. Mol. Biol. 309, 401-421. 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)

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fd Architecture

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fd (6.5 x 880 nm)

1/100th virion length

ssDNAcore

65 Å

Caspar & Makowski (1981) J. Mol. Biol. 145, 611-617.Day et al. (1988) Ann. Rev. Biophys. 17, 509-539.Marvin et al. (1994) J. Mol. Biol. 235, 260-286.

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?)

Molecular Models of the fd Capsid

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Zeri et al. (2003) PNAS 100, 6458-6463.

solid state NMR

Marvin et al. (2006) J. Mol. Biol. 355, 294-309.

fiber X-ray diffraction

Bacteriophages