Phage identification and characterization LECTURE 11: Phage identification and characterization Viro102: Bacteriophages & Phage Therapy 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB)

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

Bacteriophage: ICTV Classification Nucleic acid Morphology Family Linear dsDNA Non-enveloped, contractile tail Myoviridae Non-enveloped, long non-contractile tail Siphoviridae Non-enveloped, short non contractile tail Podoviridae Non-enveloped, isometric Tectiviridae Circular dsDNA Corticoviridae Enveloped, rod-shaped Lipothrixvirida

Bacteriophage: ICTV Classification Circular dsDNA Enveloped, pleomorphic Plasmaviridae Linear dsDNA Non-enveloped, rod-shaped Rudiviridae Non-enveloped, lemon-shaped Fuselloviridae Circular ssDNA Non-enveloped, filamentous Inoviridae Non-enveloped, isometric Microviridae Linear ssRNA Leviviridae Segmented dsRNA Enveloped, spherical Cystoviridae

Bacteriophage Characterization Electron microscopy (Morphological Studies) Bacteriophage DNA isolation RFLP Genome fingerprinting by RAPD analysis SDS-PAGE analysis of phage proteins Burst size Bacteriophage host range

Electron Microscopy Aliquots of a bacteriophage sample obtained by ultracentrifugation were subjected to electron microscopy for morphological analysis. Purified phage particles were negatively stained with 2% (wt/vol) uranyl acetate, deposited on carbon-coated grids.

Isometric heads, visible collars, and shorter contractile tails with terminal base plates, which are characteristics of the family Myoviridae Electron micrographs of L. fallax bacteriophages. (A) R01; (B) R03; (C) R05; (D) R09; (E) R12; (F) R19. Bars

Electron Microscopy

Electron Microscopy

Bacteriophage host range Phage host range can be done by using the following test method against different host Spot test Plaque assay Streak Assay

Bacteriophage host range Spotting can be used to provide a first approximation of the ability of a phage to lyse or just kill a bacterial strain. This is done such as during phage typing procedures or, at lower phage densities, as a means of phage titering that is less materials intensive than full plate-count assays.  Spotting can be used as a means of determining a phage's host range. Spot Test

Bacteriophage host range Streak Assay

Plaque counting Unit (PFU) virulent phage stock and a susceptible host cell culture. 10-fold dilutions of the phage stock are prepared. The procedure requires the use of a Double-Layer Agar (DLA) technique also known as double agar overlay method, in which the hard agar serves as a base layer (to form gel), and a mixture of few phage particles (diluted stock) and a very large number of host cells in a soft agar forms the upper overlay. When the plates are incubated, susceptible E. coli cells multiply rapidly and produce a lawn of confluent growth on the medium. When one phage particle adsorbs to a susceptible cell, penetrates the cell, replicates and release new phage particles which infect other bacteria in the vicinity of the initial host cell.

The destroyed cells produce single circular, non turbid areas called plaques in the bacterial lawn, where there is no growth of bacteria. Each plaque represents the lysis of a phage-infected bacterial culture and can be designated as a plaque-forming unit (PFU) and is used to quantitate the number of infective phage particles in the culture.  Dyes that stain the living cells are frequently used to enhance the contrast between the plaques and the living cells

Bacteriophage DNA isolation One hundred ml of phage lysate was incubated for 1 h at 37°C after addition of DNase I and RNase A(2 µg/ml). Incubated The phages were pellet down by centrifugation phage pellets To degrade bacterial DNA and RNA in the lysate, you will use a mix of nucleases (DNase I and RNase A). The capsid of the phage protects its DNA from the nucleases. However, if nucleases are present in later steps of the protocol, the phage DNA may become degraded.

Phenol Chloroform extraction The pellet obtained after centrifugation is dissolved in 100 micro liter autoclaved H20. Treated with DNase I & incubated for 370C SDS, proteinase K Phenol chloroform extraction

Phenol chloroform method

Phage Genome (Agarose Gel) 23130bp 9416bp 6557bp 4361bp 2322bp 2027bp 1 2 3 4 20-23 kb

Genome fingerprinting by restriction fragment length polymorphism (RFLP) analysis Purified bacteriophage DNA samples were subjected to restriction enzyme digestion with AluI, BamHI, EcoRI, HindIII, MboI, RsaI, and Sau3AI The restriction digests were separated on a 0.8% agarose gel and stained with ethidium bromide.

RFLP analysis of phage DNA: EcoRI digestion patterns of Myoviridae bacteriophages. Lane M, 1-kb DNA ladder; lane 1, R03; lane 2, R05; lane 3, R12.

Genome fingerprinting by RAPD analysis The method used for randomly amplified polymorphic DNA (RAPD) analysis Add primers and template DNA PCR Agarose gel

Phage A Phage B

SDS-PAGE analysis of phage proteins. Bacteriophage structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Crude extract Specific protein extraction

SDS-PAGE patterns of phage structural proteins SDS-PAGE patterns of phage structural proteins. Lanes M, molecular weight markers; lane 1, Siphoviridae phage R01; lane 2, Myoviridae phage R03; lane 3, Siphoviridae phage R09.

Phage Burst Size, Latent period 801 LK1/ cell 24 min. One step growth curve showing the latent period (24 min) and the average burst size (801 viral particles per host cell). Latent time and burst size of phage LK1 were inferred from the curve with a triphasic pattern. L: latent phase;R: rise phase; P: plateau phase.

Thanks!! Measuring bacteriophage burst size Does each phage-infected bacterial cell release the same number of phage or does the number vary from cell to cell?   If it varies, can we define the factors involved in this variation?  To measure an average burst size lets perform this online experiment http://virtuallaboratory.colorado.edu/BioFun-Support/labs/OnMutation/section_04.html http://virtuallaboratory.colorado.edu/BioFun-Support/labs/OnMutation/flash/vCOUNT.html Thanks!!