Bacteriophages PDF

Bacteriophages Chapter 6 Notice: This material is subject to the U.S. Copyright Law; further reproduction in violation of the law is prohibited. Image © 2020 McGenity et al. Microbial Biotechnology published by John Wiley & Sons Ltd and So...

Bacteriophages Chapter 6 Notice: This material is subject to the U.S. Copyright Law; further reproduction in violation of the law is prohibited. Image © 2020 McGenity et al. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. CC BY 4.0 Viruses Viruses are genetic information (DNA or RNA) contained within a protective protein coat Straddle the definition of life (= capacity for growth, reproduction, functional activity) outside of host: Inactive - Cannot reproduce outside of living cells. Inside: direct activities of the cell to replicate itself Thus, Obligate Intracellular Parasite Infectious agents, NOT microorganisms © 2020 McGenity et al. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. CC BY 4.0 Bacteriophage ▪ Viruses typically infect specific hosts and a range of cells within these hosts – viral tropism An example of a human virus is the influenza virus. An example of a plant virus is the tobacco mosaic virus (first virus discovered). Cause $$$$$ in crop damage A bacteriophage ( or phage) is a virus that infects bacteria. A coliphage infects E. coli © 2020 McGenity et al. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. CC BY 4.0  Viruses Infect Specific Hosts Most viruses infect only specific types of cells in one host Ex: HIV -> Human Helper T-Cells (CD4) Host range is determined by interactions between viral (spike) and host surface molecules (receptor) © McGraw Hill, LLC  FDA approved use of species-specific bacteriophages to control food-contaminating bacteria (Salmonella, E. coli); May provide alternative to antibiotics Application of a Phage Cocktail for Control of Salmonella in Foods and Reducing Biofilms Islam MS, Zhou Y, Liang L, Nime I, Liu K, Yan T, Wang X, Li J. Application of a Phage Cocktail for Control of Salmonella in Foods and Reducing Biofilms. Viruses. 2019; 11(9):841. https://doi.org/10.3390/v1109 0841 Licensee MDPI, Basel, Switzerland. (CC BY) license (http://creativecommons.org/li censes/by/4.0/).  Phage Therapy Biospace.com “Balasa took part in a procedure at Yale University that used bacteriophages that attack and kill P. aeruginosa. It was a last-ditch effort to avoid a high-risk lung transplant. She was the eighth patient to try the approach.” Vaitekenas A, Tai AS, Ramsay JP, Stick SM, Kicic A. Pseudomonas aeruginosa Resistance to Bacteriophages and Its Prevention by Strategic Therapeutic Cocktail Formulation. Antibiotics. 2021; 10(2):145. https://doi.org/10.3390/antibiotics10020145 Licensee MDPI, Basel, Switzerland. CC BY 4.0  Ecological Roles of Viruses ▪ Viruses play vital roles in ecosystems. ▪ Virome - The sum of viral populations in an ecosystem ▪ Viruses limit host population density by acting as predators/parasites. Regulate microbiomes - Bacteriophages in human gut regulate bacterial populations. Host death recycles nutrients back to the community. Zuppi M, Hendrickson HL, O'Sullivan JM, Vatanen T. Phages in the Gut Ecosystem. Frontiers in Cellular and Infection Microbiology. 2021 ;11:822562. DOI: 10.3389/fcimb.2021.822562. PMID: 35059329; PMCID: PMC8764184. Copyright © 2022 Zuppi, Hendrickson, O’Sullivan and Vatanen. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY).  Ecological Roles of Viruses ▪ Viruses limit host population density by acting as predators/parasites. Regulate microbiomes - Bacteriophages in human gut regulate bacterial populations. Host death recycles nutrients back to the community. Gao Y, Lu Y, Dungait JAJ, Liu JB, Lin SH, Jia JJ and Yu GR (2022) The “Regulator” Function of Viruses on Ecosystem Carbon Cycling in the Anthropocene. Front. Public Health 10:858615. doi: 10.3389/fpubh.2022.858615. Copyright © 2022 Gao, Lu, Dungait, Liu, Lin, Jia and Yu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). General Characteristics of Viruses Most viruses notable for small size 100-1,000 times smaller than cells they infect – need electron microscope © McGraw Hill, LLC Smallest: ~10 nm ~10 genes (~1800 bp) ~Porcine circovirus Largest: ~800 nm Mimiviruses (amoeba virus) Light microscope Size of smallest bacteria Once thought to be a bacterium Publication: ViralZone: a knowledge resource to understand virus diversity. Hulo C, de Castro E, Masson P, Bougueleret L, Bairoch A, Xenarios I, Le Mercier P. Nucleic Acids Res. 2011 Jan;39(Database issue):D576-82. CC BY General Characteristics of Viruses Virion (viral particle) consists of nucleic acid & protein coat Protein coat is called a capsid: protects viral nucleic acids Composed of identical protein subunits called capsomeres/ protomers Embedded with required proteins for infection called spikes Capsid + nucleic acid = nucleocapsid Enveloped viruses have a lipid bilayer (envelope) that is obtained when leaving host cell More susceptible to disinfectants & hand sanitizer, may help evade host immune system Matrix protein between nucleocapsid and envelope Non-enveloped viruses lack envelope Parker et al. 2022 OpenStax Microbiology CC BY 4.0. General Characteristics of Viruses Cloaked viruses Hepatitis A virus non-enveloped in environment, not within host hepatitis A virions are released from cells in membrane vesicles containing 1-4 virus particles Viral envelopes typically contain viral glycoproteins – Spikes Aid in attachment to cell receptors The target of antibodies that block viral infection CREDIT: MEDICAL ARTS, NIH PUBLIC DOMAIN The presence of an envelope without spikes helps HAV evade immune cells General Characteristics of Viruses ▪ Viruses have protein components for attachment Many eukaryotic viruses have spikes Phages have tail fibers Allow virion to interact and attach to specific receptor sites on host cell Ami Images/Science Source © McGraw Hill, LLC Three main shapes: Helical/Filamentous Appear cylindrical Capsomeres arranged in a helix Icosahedral 20 triangles Complex Tailed - Icosahedral head connected to protein tail – Most phages Asymetrical - characteristics of icosahedral and helical viruses © McGraw Hill, LLC  Helical Capsids Shaped like hollow tubes with protein walls. Protomers self assemble into rigid tube. Size of capsid is influenced by promoters and genome. (a) Photo Researchers/Science History Images/Alamy Stock Photo © McGraw Hill, LLC  Icosahedral Capsids Most efficient way to enclose a space. An icosahedron is a regular polyhedron with 20 equilateral triangular faces and 12 vertices. Capsomers—Ring or knob- shaped units made of 5 or 6 protomers. Biophoto Associates/Science Source © McGraw Hill, LLC  Capsids of Complex Symmetry Some viruses do not fit into the category of having helical or icosahedral capsids. Poxviruses—largest of the animal viruses. Complex interior and ovoid- to brick-shaped exterior. Large bacteriophages—binal symmetry (head resembles icosahedral, tail is helical). © McGraw Hill, LLC Viral Genomes ▪ Viral genomes can be: DNA or RNA © McGraw Hill, LLC 4,000 nucleotides to 2 million nucleotides genes may overlap in sequence Single- or double-stranded (ss or ds) Linear, circular, or segmented Image of viral genome. Pavesi, A. Origin, Evolution and Stability of Overlapping Genes in Viruses: A Systematic Review. Genes 2021, 12, 809. https://doi.org/10.3390/genes12060809 © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).  General Characteristics of Viruses ▪ Virus families end in suffix -viridae Some indicate appearance (ex: Coronaviridae from corona, meaning “crown”) Others named for geographic area from which first isolated (ex: Bunyaviridae from Bunyamwera in Family: Picornaviridae Uganda, Africa) Genus: Enterovirus ▪ Genus ends in -virus (for example, Ebolavirus) Species: Enterovirus C ▪ Species/subtype name often name of disease Subtype: Poliovirus For example, poliovirus causes polio Unlike bacteria, viruses are commonly referred to only by species name  General Characteristics of Viruses Viruses often referred to and grouped informally *Disease-causing viruses often grouped by transmission route* Enteric viruses - Generally transmitted via fecal-oral route - Ex: norovirus, rotavirus - Often cause “stomach flu” (gastroenteritis) ‣ Some can cause systemic disease Respiratory viruses - Usually inhaled via infected respiratory droplets - Generally, remain localized in respiratory tract - Ex: Influenza, rhinovirus (common cold), coronaviruses Zoonotic viruses - Transmitted from non-human animal to human via arthropod vector (arbovirus) or direct contact - Ex: West Nile virus, rabies virus  There are an estimated 1031 bacteriophages on Earth, more than any Bacteriophages other biological entity ▪ Bacteriophages infect bacterial cells ▪ Three general types characterized based on relationship with host 1. Lytic phages 2. Temperate phages Phages destroy up to 40% of 3. Filamentous phages the bacteria in Earth's oceans each day Image 1 Powledge TM (2004) New Antibiotics—Resistance Is Futile. PLoS Biol 2(2): e53. https://doi.org/10.1371/journal.pbio.0020053 Photograph courtesy of Vincent Fischetti and Raymond Schuch, The Rockefeller University CC BY Image 2"Reef edge at Halahi Reef, Red Sea, Egypt #SCUBA #UNDERWATER #PICTURES" by Derek Keats is licensed under CC BY 2.0. Lytic Phage Infections Lytic/virulent phages replicate using host machinery and new viral particles exit host by lysing the cell Lytic phages are classified as an acute virus, yield productive infections – production of virions occurs immediately (vs latent infection) T4 phage (dsDNA) as model; entire process takes ~30 minutes in 5 step process 1. Attachment 2. Genome entry 3. Biosynthesis 4. Assembly/Maturation 5. Release/Lysis Parker et al. 2022 OpenStax Microbiology CC BY 4.0. Lytic Phage Infections 1. Attachment Phage collides with host cell ( T4 = E. coli) by chance Viral tail fiber binds to host cell receptor (specific for each virus) Host cell receptor usually a pilus or other cell surface structure (T4 = E. coli LPS & membrane protein C) Any cell that lacks the specific receptor are resistant Host bacteria can evolve resistance to phage attachment via masking or mutation of surface receptors. Chaturongakul S and Ounjai P (2014) Phage–host interplay: examples from tailed phages and Gram-negative bacterial pathogens. Front. Microbiol. 5:442. doi: 10.3389/fmicb.2014.00442 © 2014 Chaturongakul and Ounjai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Lytic Phage Infections 2. Genome entry T4 lysozyme (located in the baseplate) degrades peptidoglycan (bond btw NAG and NAM) Tail sheath contracts and injects genome through cell wall and membranes The phage capsid (now termed a “ghost”) remains outside, attached to the cell surface. © McGraw Hill, LLC  Lytic Phage Infections 3. Synthesis Phage DNA is transcribed and translated into proteins by host machinery Not all T4 proteins are synthesized simultaneously Early proteins translated within minutes from viral DNA prevent host gene expression DNA-dependent DNA polymerase Nucleases degrades host DNA/RNA Proteins that modify host RNA polymerase so that it no longer recognizes bacterial promoters Already present bacterial enzymes continue to function (biosynthesis and energy harvesting) to replicate viral genome and make viral proteins Late proteins are structural proteins produced toward end of cycle Capsomeres=capsid, tail proteins, tail fibers By Anne Chevallereau and colleagues at https://www.eurekalert.org/pub_releases/2016-07/p-ppt070116.php - https://www.eurekalert.org/multimedia/pub/web/118913_web.jpg at https://www.eurekalert.org/multimedia/pub/118913.php, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=99660965 Lytic Phage Infections 4. Assembly (maturation) Once parts are produced, they assemble Some components spontaneously assemble, others require protein scaffolds Multi-step sequence: 1. Head (capsid) formed and packed with genome 2. Tail formed and attached to head 3. Tail spikes/fibers are attached Source: Aksyuk AA, Rossmann MG. Bacteriophage Assembly. Viruses. 2011; 3(3):172-203. https://doi.org/10.3390/v3030172 CC BY  Lytic Phage Infections 5. Release Proteins produced late in infection Holin creates holes in the plasma membrane T4 lysozyme (Endolysin) degrades peptidoglycan Cell lyses, releases phages Burst size (number of virions released) of T4 is ~200 Image by Provided by James L. Van Etten, Irina V. Agarkova, David D. Dunigan. Photographer(s): Meints et al. - https://www.mdpi.com/viruses/viruses-12-00020/article_deploy/html/images/viruses-12-00020-g001.png at https://www.mdpi.com/1999- 4915/12/1/20/htm (extract)Viruses 2020, 12(1), 20;doi:10.3390/v12010020This article belongs to the Special Issue Viruses Ten-Year Anniversary. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=101355038 Lytic Phage Infections Total process takes ~25 minutes © McGraw Hill, LLC Bacteriophages ▪ Temperate Phages have 2 options: produce a 1. Lytic infection 2. Lysogenic infection -incorporate DNA into host cell genome Phage DNA that incorporates into host chromosome is called a prophage Prophage is replicated along with host DNA during binary fission © McGraw Hill, LLC Temperate Phages Decision between lysogenic or lytic cycles appears to be random However, dictated by environmental cues and host cell factors that either activate or repress transcription of genes for prophage excision Influenced by… Metabolic state If cell growing slowly (limited nutrients) than lysogenic infection is more likely Wait until better environment, more cells to infect Number of host cells present If low host cell pop., lysogenic better Events that threaten host cell survival (uv-light)  Viruses can communicate to direct lytic vs. lysogenic cycle (2017) “Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis–lysogeny decisions During infection of its Bacillus host cell” Erez, Z., Steinberger-Levy, I., Shamir, M. et al. Communication between viruses guides lysis–lysogeny decisions. Nature 541, 488–493 (2017). https://doi.org/10.1038/nature21049 Virus communication Phage begins with lytic infection Lysogenic cycle is inhibited at the first encounter of a phage with a bacterial population aimR and aimP are expressed immediately upon infection aimR protein activates (activator) AimX expression AimX transcripts prevents gene expression of lysogenic proteins This results in a lytic cycle Meanwhile, AimP is expressed, translated, and secreted This gene makes a communication peptide Erez, Z., Steinberger-Levy, I., Shamir, M. et al. Communication between viruses guides lysis–lysogeny decisions. Nature 541, 488–493 (2017). https://doi.org/10.1038/nature21049 called arbitrium Virus communication At later stages of the infection dynamics The arbitrium peptide accumulates in environment Is internalized into the bacteria by a transporter – OPP. Now, when a phage infects a new bacterium Arbitrium molecules binds to AimR activator. AimR cannot activate the expression of AimX (repressor of lysogenic genes), leading to lysogeny. Erez, Z., Steinberger-Levy, I., Shamir, M. et al. Communication between viruses guides lysis–lysogeny decisions. Nature 541, 488–493 (2017). https://doi.org/10.1038/nature21049 Virus communication But why? Allows virus to coordinate their attack At the beginning of infection, it makes sense for the viruses to quickly replicate (lytic cycle) - large bacteria population If they don’t switch strategies, there won't be any hosts left for future generations of viruses to infect At some point, the viruses need to switch strategies and become dormant so that the bacterial population can recover Erez, Z., Steinberger-Levy, I., Shamir, M. et al. Communication between viruses guides lysis–lysogeny decisions. Nature 541, 488–493 (2017). https://doi.org/10.1038/nature21049  Temperate Phage Infections ▪ Lambda (λ) phage as model Phage lambda binds to Escherichia coli by contact between its tail tip (J protein) and the maltose porin (LamB gene) The DNA is then threaded into the cytoplasm via an extrusion mechanism Linear chromosome; cohesive ends chromosome anneal and are ligated, circularizing the genome in the host © McGraw Hill, LLC Phage image By Sankohm at English Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=41398886  Temperate Phage Infections ▪ Lambda (λ) phage as model Resulting circular molecule either directs lytic infection or integrates into E. coli chromosome Regulatory Proteins Determine Lysogeny or the Lytic Cycle Function as repressors, activators, or both. cII activator plays a role in determining if λ will establish lysogeny or follow the lytic cycle. cII levels high early in infection—lysogeny. Integrase—catalyzes integration of lambda genome into host chromosome. cII levels not high early in infection—lytic cycle. © McGraw Hill, LLC Phage Infection Now, the host RNA polymerase can begin to transcribe the phage operons. At first, though, just a few key proteins are expressed, most notably the “control proteins” Cro and CI. Cro protein leads to lysis. Small amounts of Cro activate the lytic cycle. CI protein (lambda repressor) leads to lysogeny. Citation: Dorri F, Mahini H, Sharifi-Zarchi A, Totonchi M, Tusserkani R, Pezeshk H, et al. (2014) Natural Biased Coin Encoded in the Genome Determines Cell Strategy. PLoS ONE 9(8): e103569. https://doi.org/10.1371/journal.pone.0103569 © 2014 Dorri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, By Chimb - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=30012718  Phage Infection What determines who wins? Nutrient availability: In the gut between meals, nutrient levels are low, E. coli protease HflB is inhibited from cleaving the phage repressor Mutations/UV Damage: RecA levels increase and the protease destroys phage repressor protein (CI) Phage population density: When multiple phages infect the same cell, phage repressor expression increases Top image by Vhyr Qhan - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=29750564 Bottom image (modified) by Minsik Kim et al. PNAS May 3, 2016 113 (18) E2480-E2488 CC BY Temperate Phage Infections ▪ Lambda (λ) phage as model λ Phage can use enzyme integrase to insert itself (DNA) at specific host site – now called prophage Replicates with host chromosome CI (Phage repressor protein) transcribed during the lysogenic state which maintains lysogeny and prevents superinfection by other phage lambda particles. CI also represses the expression of Cro and proteins of the lytic cycle. The levels of CI are decreased by host RecA, which cleaves this protein along with many host repressors – Cro -> induction -> lytic infection RecA is activated by DNA damage, so UV light can induce lysis in 100% of a lysogenic population. By Chimb - Microsoft Powerpoint, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=30012672 Temperate Phage Infections Lambda (λ) phage DNA excised from chromosome only about 1 out of 10,000 host cell divisions If DNA damaged (ex: UV light exposure), SOS repair system turns on, activates a protease (RecA) RecA destroys phage repressor protein (CI) responsible for keeping prophage in chromosome, allows expression of lytic genes, enter lytic cycle Minsik Kim et al. PNAS May 3, 2016 113 (18) E2480-E2488 CC BY Temperate Phage Infections Lysogen (infected cell) is morphologically identical to an uninfected cell, but other aspects may change: Immunity to superinfection: lysogens protected against infection by same phage Phage repressor protein binds to operator of incoming phage DNA (represses Cro) Prevents expression of genes that direct lytic infection Lysogenic conversion: prophage changes phenotype of lysogen Often toxins are encoded by genes on prophage (ex: botulinum toxin) Only strains carrying prophage produce the toxins Modification of image in Minsik Kim et al. PNAS May 3, 2016 113 (18) E2480-E2488 CC BY  Bacteriophages Filamentous Phages Single-stranded DNA phages Look like long fibers Do NOT cause lytic infections Host cells not killed (lysed), but grow slower while virions are produced, secreted Still classified as a productive infection (vs. latent) M13 phage as model Attaches to F pilus of E. coli Publication: ViralZone: a knowledge resource to understand Single-stranded DNA genome virus diversity. Hulo C, de Castro E, Masson P, Bougueleret L, Bairoch A, Xenarios I, Le Mercier P. Nucleic Acids Res. 2011 Jan;39(Database issue):D576-82. CC BY enters the cytoplasm Filamentous Phages Protein Synthesis and Release Viral proteins form pores that span from cytoplasmic membrane to the outer membrane M13 phage capsomeres synthesized and inserted into cytoplasmic membrane As phage DNA excreted through pores, capsomeres coat the DNA, form nucleocapsid – process called Extrusion This all occurs as the cell is dividing so progeny of host cell also are infected (carrier cells) Publication: ViralZone: a knowledge resource to understand virus diversity. Hulo C, de Castro E, Masson P, Bougueleret L, Bairoch A, Xenarios I, Le Mercier P. Nucleic Acids Res. 2011 Jan;39(Database issue):D576-82. CC BY The Roles of Bacteriophages in Horizontal Gene Transfer Phages can accidently transfer bacterial DNA from one bacteria (donor) to another (recipient) Called transduction – two types: 1. Generalized transduction 2. Specialized transduction The Roles of Bacteriophages in Horizontal Gene Transfer Generalized Transduction Results from packaging error during phage assembly Lytic and temperate phages degrade host chromosome via nuclease Host DNA fragments mistakenly packaged into phage head Termed generalized transducing particles Following release, can bind to new host, inject DNA DNA may integrate via homologous recombination, replacing host DNA Any gene from donor cell can be transferred © McGraw Hill, LLC The Roles of Bacteriophages in Horizontal Gene Transfer Specialized Transduction Excision mistake during induction (transition from lysogenic to lytic) of temperate phage Short piece of flanking bacterial DNA accidently removed; piece of phage DNA remains Excised DNA incorporated into phage heads; defective transducing particles released Can bind to new host, inject DNA Bacterial DNA may integrate via homologous recombination or via intergrase Only bacterial genes adjacent to prophage transferred © McGraw Hill, LLC Bacterial Defenses ▪ Bacteria have evolved several forms of defense against bacteriophage infection: Mask receptor proteins – genetic resistance -> mutations -> Altered receptor proteins – Cover receptors Restriction endonucleases – Cleave viral DNA lacking methylation CRISPR integration of phage DNA sequences – Clustered regularly interspaced short palindromic repeats "Fi le:12 Hegasy Cas9 Immun Wiki E CCBYSA.png" by Guido4 is licensed under CC BY-SA 4.0. – A bacterial immune system Bacterial Defenses Against Phages Preventing Phage Attachment Alter or cover specific receptors on the cell surface Mutations may lead to altered surface receptors that the virus does not recognize. Capsules, slime layers, biofilms mask receptors Staphylococcus aureus produces protein A, which covers phage receptors © 2019 Beata Orzechowska and Manal Mohammed. Licensee IntechOpen. DOI: 10.5772/intechopen.87247 This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License.  Bacterial Defenses Against Phages Restriction-Modification Systems – protect bacteria from phage infection by quickly degrading foreign DNA Requires two enzymes: 1. Restriction enzymes recognize short nucleotide sequences (incoming phage DNA) and cut DNA at a specific site Bacteria have hundreds of varieties, each recognizing different sequences 2. Modification enzymes methylate recognition site on bacterial DNA so not attacked by their own restriction enzymes Enzymes may accidentally methylate phage DNA - allowing infection © 2019 Beata Orzechowska and Manal Mohammed. Licensee IntechOpen. DOI: 10.5772/intechopen.87247 This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License. Bacterial Defenses Against Phages CRISPR system Cells that survive phage infections insert pieces of phage DNA (spacer DNA) into region of DNA called CRISPR Provides record of infection CRISPR region transcribed, cut into small pieces called crRNAs (Guide RNA) crRNAs bind to Cas proteins When injected phage DNA Binds to CAS-crRNA complex it is targeted for destruction Some phages have anti-CRISPR gene that codes for the protein Acr, which inhibits Cas proteins "Fi le:12 Hegasy Cas9 Immun Wiki E CCBYSA.png" by Guido4 is licensed under CC BY-SA 4.0.  Methods Used to Study Bacteriophages ▪ Viruses multiply only inside living cells Must cultivate suitable host cells to grow viruses Plaque assays used to quantify phage particles in samples: sewage, seawater, soil Agar inoculated with bacterial host and filtered sample (remove bacteria) Bacterial lawn forms Zones of clearing form from infected cells lysing, called plaques Counting plaque forming units (PFU) yields titer – conc. of infectious phage in original sample Difficult/Impossible with temperate & filamentous phage © McGraw Hill, LLC Methods Used to Study Bacteriophages Methods Used to Study Bacteriophages Plaque assays Dilutions of virus samples made and plated with appropriate host cells. Number of plaques counted. Results expressed as plaque-forming units (PFU). Directly proportional to number of viruses. © McGraw Hill, LLC